Programming Amphiphilic Peptoid Oligomers for Hierarchical Assembly and Inorganic Crystallization.
Cai Bin,Li Zhiliang,Chen Chun-Long
Accounts of chemical research
ConspectusNatural organisms make a wide variety of exquisitely complex, nano-, micro-, and macroscale structured materials in an energy-efficient and highly reproducible manner. During these processes, the information-carrying biomolecules (e.g., proteins, peptides, and carbohydrates) enable (1) hierarchical organization to assemble scaffold materials and execute high-level functions and (2) exquisite control over inorganic materials synthesis, generating biominerals whose properties are optimized for their functions. Inspired by nature, significant efforts have been devoted to developing functional materials that can rival those natural molecules by mimicking in vivo functions using engineered proteins, peptides, DNAs, sequence-defined synthetic molecules (e.g., peptoids), and other biomimetic polymers. Among them, peptoids, a new type of synthetic mimetics of peptides and proteins, have received particular attention because they combine the merits of both synthetic polymers (e.g., high chemical stability and efficient synthesis) and biomolecules (e.g., sequence programmability and biocompatibility). The lack of both chirality and hydrogen bonds in their backbone results in a highly designable peptoid-based system with reduced structural complexity and side chain-chemistry-dominated properties.In this Account, we present our recent efforts in this field by programming amphiphilic peptoid sequences for (1) the controlled self-assembly into different hierarchically structured nanomaterials with favorable properties and (2) manipulating inorganic (nano)crystal nucleation, growth, and assembly into superstructures. First, we designed a series of amphiphilic peptoids with controlled side chain chemistries that self-assembled into 1D highly stiff and dynamic nanotubes, 2D membrane-mimetic nanosheets, hexagonally patterned nanoribbons, and 3D nanoflowers. These crystalline nanostructures exhibited sequence-dependent properties and showed promise for different applications. The corresponding peptoid self-assembly pathways and mechanisms were also investigated by leveraging in situ atomic force microscopy studies and molecular dynamics simulations, which showed precise sequence dependency. Second, inspired by peptide- and protein-controlled formation of hierarchical inorganic nanostructures in nature, we developed peptoid-based biomimetic approaches for controlled synthesis of inorganic materials (e.g., noble metals and calcite), in which we took advantage of the substantial side chain chemistry of peptoids and investigated the relationship between the peptoid sequences and the morphology and growth kinetics of inorganic materials. For example, to overcome the challenges (e.g., complexity of protein- and peptide-folding, poor thermal and chemical stabilities) facing the area of protein- and peptide-controlled synthesis of inorganic materials, we recently reported the design of sequence-defined peptoids for controlled synthesis of highly branched plasmonic gold particles. Moreover, we developed a rule of thumb for designing peptoids that predictively enabled the morphological evolution from spherical to coral-shaped gold nanoparticles (NPs). With this Account, we hope to stimulate the research interest of chemists and materials scientists and promote the predictive synthesis of functional and robust materials through the design of sequence-defined synthetic molecules.
β-tripeptides act as sticky ends to self-assemble into a bioscaffold.
Del Borgo Mark P,Kulkarni Ketav,Tonta Mary A,Ratcliffe Jessie L,Seoudi Rania,Mechler Adam I,Perlmutter Patrick,Parkington Helena C,Aguilar Marie-Isabel
Peptides comprised entirely of β-amino acids, commonly referred to as β-foldamers, have been shown to self-assemble into a range of materials. Previously, β-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the β-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two β-tripeptides. These hybrid β-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a β-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that β-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold.
Cytocompatibility of self-assembled beta-hairpin peptide hydrogel surfaces.
Kretsinger Juliana K,Haines Lisa A,Ozbas Bulent,Pochan Darrin J,Schneider Joel P
MAX1 is a 20 amino acid peptide that undergoes triggered self-assembly to form a rigid hydrogel. When dissolved in aqueous solutions, this peptide exists in an ensemble of random coil conformations rendering it fully soluble. The addition of an exogenous stimulus results in peptide folding into beta-hairpin conformation. This folded structure undergoes rapid assembly into a highly crosslinked hydrogel network. DMEM cell culture media is one stimulus able to initiate folding and consequent self-assembly of MAX1. The cytocompatibility of this gel towards NIH 3T3 murine fibroblasts is demonstrated. Gels were shown to be non-toxic to the fibroblast cells. MAX1 hydrogels also foster the ability of the cells to attach to the hydrogel scaffold in the absence or presence of serum proteins. Additionally MAX1 hydrogels were able to support fibroblast proliferation to confluency with little effect on the rheological properties of the scaffold. MAX1 hydrogels meet the preliminary mechanical and cytocompatibiltiy requirements of a tissue engineering scaffold.
Self-assembled octapeptide scaffolds for in vitro chondrocyte culture.
Mujeeb Ayeesha,Miller Aline F,Saiani Alberto,Gough Julie E
Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.
Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells.
Zhou Mi,Smith Andrew M,Das Apurba K,Hodson Nigel W,Collins Richard F,Ulijn Rein V,Gough Julie E
We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.
Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization.
Abdali Zahra,Aminzare Masoud,Zhu Xiaodan,DeBenedictis Elizabeth,Xie Oliver,Keten Sinan,Dorval Courchesne Noémie-Manuelle
ACS synthetic biology
Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling β-helical subunit composed of five pseudorepeats (R1-R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphology of the truncated and HAP-containing curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocrystalline HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.
Bioactive gel self-assembled from phosphorylate biomimetic peptide: A potential scaffold for enhanced osteogenesis.
Quan Changyun,Zhang Zhaoqing,Liang Peiqing,Zheng Junjiong,Wang Jiping,Hou Yulin,Tang Qiyan
International journal of biological macromolecules
Bone morphogenetic protein-2 biomimetic peptide (BMPBP) is a potent osteoinductive cytokine and plays a critical role during bone regeneration. Efforts to prepare hydrogels with surface modification or physical absorption of bioactive molecules do not provide sufficient bioactivity to meet the requirements of clinical application. The goal of this study was to form a three-dimensional hydrogel comprised of BMP-2 core sequence oligopeptide, phosphoserine, a synthetic cell adhesion peptide (RGDS), and polyaspartic acid to synergistically promote osteogenesis. Experiments performed in vitro revealed that the peptide gel was conducive to adhesion and proliferation of rat marrow mesenchymal stem cells (rMSCs). In addition, RT-PCR analysis indicated that rMSCs allowed better expression of osteogenesis-related genes such as BMP-2, runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Use of the rat cranial bone defects model with micro-CT 3D reconstruction showed that bone regeneration patterns occurred from one side edge toward the center of the area implanted with the prepared biomimetic peptide hydrogels, demonstrating significantly accelerated bone regeneration. This work will provide a basis to explore the further application potential of this bioactive scaffold.
[NELL-1: a novel highly efficient and specific growth factor].
Qin X Y,Zhao H X,Zhang Q,Chen F,Lin J X
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences
Regeneration of bone tissue, as well as other tissues, requires involvement and interaction of cells, scaffolds and relevant growth factors, among which growth factors play a crucial role in maintaining the stability of microenvironment. Nel-like-type 1 molecule (NELL-1), a novel growth factor in tissue engineering, has been studied intensively in recent years. Researches mainly covered gene and protein structure and their expression profiling, biological function, molecular mechanisms and disease relevance. NELL-1 expressed in embryonic tissue is essential for growth and development of bone tissue. NELL-1 presents excellent abilities of inducing bone and cartilage regeneration,especially with high specificity to chondrocyte lineage. Compared with classic osteogenic growth factor bone morphogenetic protein 2 (BMP-2), the process of osteogenesis interacted with NELL-1 exhibits stronger specificity, higher bone density and fewerside effects. Furthermore, a recent study shows synergistic effects of NELL-1 and BMP-2. NELL-1 enhances the osteogenic reaction induced by BMP-2 of cells and notably declines inflammation response caused by BMP-2. This review evaluates the current research progress of the function and application of NELL-1 by the systematic method of evidence-based medicine.
Peptide-modified bone repair materials: Factors influencing osteogenic activity.
Liao Jie,Wu Shuai,Li Kun,Fan Yubo,Dunne Nicholas,Li Xiaoming
Journal of biomedical materials research. Part A
Many factors have been demonstrated as having an influencing effect on the osteogenic activity of the peptide-modified bone repair materials. However, most of studies only focus on one or two aspects that result in an incomplete direction for materials preparation, characterization, and performance evaluation. In this review, we reported several factors through summarizing previous research studies, which are mainly centered on three aspects: (1) the characteristics of peptide immobilized on the surface of matrix (e.g., type and length of sequence, structure, and density); (2) the combination mode between peptide and matrix (including covalent binding in selective or nonselective immobilization, and noncovalent binding through simple absorption or mixing with the matrix, and other factors in covalent binding); and (3) the properties of the matrix (including surface structure and morphology, dimension, mechanical properties, hydrophobic-hydrophilic balance, adsorbing proteins on materials), and the other possible influencing factors such as binding to other peptides. In addition, attentions were paid to the introduction and the discussion of newest studies and the analysis of mechanism. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.
Bioactive 3D scaffolds self-assembled from phosphorylated mimicking peptide amphiphiles to enhance osteogenesis.
Liang Peiqing,Zheng Junjiong,Zhang Zhaoqing,Hou Yulin,Wang Jiayu,Zhang Chao,Quan Changyun
Journal of biomaterials science. Polymer edition
Being an active scaffold in bone tissue engineering, hydrogel self-assembled from biomimetic peptide amphiphile (PA) has excellent ability to induce osteogenic differentiation and osteogenesis. Here, a multifunctional scaffold based on bone morphogenetic protein-2 (BMP-2) mimicking peptide, RGDS, and phosphoserine has been developed to enhance osteogenesis. Cell experiments in vitro displayed that the hydrogel could effectively promote rat messenchymal stem cells (rMSCs) proliferation and induce them differentiation into oesteblasts. The up-regulated RNA expression of osteogenic marker genes, like BMP-2, osteopontin (OPN), osteocalcin (OCN) and runt-related transcription factor 2 (Runx2) revealed that the scaffold could accelerate rMSCs differentiation at RNA level. Further studies on rat skull defect model demonstrated that the multifunctional scaffold exhibited excellent repair ability due to a potential synergistic effect of biomimetic peptide and phosphoserine. Histochemical/immunohistochemical staining results showed that expressions of alkaline phosphatase (ALP) and OCN was significantly up-regulated, indicating that the hydrogel could accelerate maturation of osteoblast precursors during the whole repairing process and be a promising bioactive scaffold for bone repairing.
[Preliminary study on the effect of vascular endothelial growth factor-loaded self-assembled peptide hydrogel on angiogenesis and vascularization of human umbilical vein endothelial cells].
Zhang R J,Wang X Z,Liu Y,Zheng S G
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology
To investigate the effect of RATEA16 scaffold on the proliferation of human umbilical vein endothelial cells (HUVEC) and the effect of new self-assembling peptide hydrogel (RATEA16) scaffold with vascular endothelial growth factor (VEGF) on promoting angiogenesis. RATEA16 hydrogel was prepared, then the injectability, microstructure, degradation, biocompatibility of RATEA16 hydrogel were determined. HUVEC were cultured with RATEA16 scaffold to detect cell morphology and proliferation. HUVEC were cultured on RATEA16 scaffold with VEGF for 24 h. The expression of VEGF-A, von Willebrand factor (vWF), matrix metalloproteinase-9 (MMP-9) and platelet endothelial cell adhesion molecule-1 (PECAM-1) were detected by using real-time PCR to evaluate the effects of the scaffold with VEGF system on HUVEC differentiation. The sol-gel transition was completed under neutral condition (pH=7.4) adjusted by Tris-HCl solution. The hydrogel could be easily injected from a syringe. It presented a porous and interconnected internal structure and the porosity of the scaffold was (67.3±9.4)%. After 4 week degradation , the residual weight was still (82.354±0.006)%, which exhibited slow degradation. HUVEC grew well after being cultured in leach liquor of RATEA16 hydrogel for 24 h, and there was no significant difference in HUVEC cell viability compared with that of the control group (>0.05). HUVEC encapsulated in RATEA16 hydrogel appeared round in shape and exhibited effectively continuous proliferation. When HUVEC were cultured on RATEA16 hydrogel with VEGF for 24 h, the formation of vascular-like structures was observed. The expression of VEGF-A and MMP-9 was 1.5-2.0 times that of control group, and vWF was 10 times and PECAM-1 was 55 times compared with that of the control group (<0.05). The RATEA16 hydrogel used in this study could be prepared by simply adjusting pH to neutral. This hydrogel exhibited good biodegradability, slow degradation and injectability. HUVEC might attach and spread in RATEA16 scaffold. The RATEA16 scaffold with VEGF could promote angiogenic differentiation of HUVEC. The novel scaffold is expected to achieve the critical vascularization process in bone tissue regeneration.
Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.
Onak Günnur,Gökmen Oğuzhan,Yaralı Ziyşan Buse,Karaman Ozan
Journal of tissue engineering and regenerative medicine
Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.
Precisely Controlled Delivery of Abaloparatide through Injectable Hydrogel to Promote Bone Regeneration.
Ning Ziyu,Tan Baoyu,Chen Bo,Lau Dzi Shing Aaron,Wong Tak Man,Sun Tianhao,Peng Songlin,Li Zhaoyang,Lu William Weijia
Side-effects from allograft, limited bone stock, and site morbidity from autograft are the major challenges to traditional bone defect treatments. With the advance of tissue engineering, hydrogel injection therapy is introduced as an alternative treatment. Therapeutic drugs and growth factors can be carried by hydrogels and delivered to patients. Abaloparatide, as an analog of human recombinant parathyroid hormone protein (PTHrp) and an alternative to teriparatide, has been considered as a drug for treating postmenopausal osteoporosis since 2017. Since only limited cases of receiving abaloparatide with polymeric scaffolds have been reported, the effects of abaloparatide on pre-osteoblast MC3T3-E1 are investigated in this study. It is found that in vitro abaloparatide treatment can promote pre-osteoblast MC3T3-E1 cells' viability, differentiation, and mineralization significantly. For the drug delivery system, 3D porous structure of the methacrylated gelatin (GelMA) hydrogel is found effective for prolonging the release of abaloparatide (more than 10 days). Therefore, injectable photo-crosslinked GelMA hydrogel is used in this study to prolong the release of abaloparatide and to promote healing of defected bones in rats. Overall, data collected in this study show no contradiction and imply that Abaloparatide-loaded GelMA hydrogel is effective in stimulating bone regeneration.
Evaluation of teriparatide effect on healing of autografted mandibular defects in rats.
Zandi Mohammad,Dehghan Arash,Gheysari Faezeh,Rezaeian Leila,Mohammad Gholi Mezerji Naser
Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery
OBJECTIVE:To evaluate the effects of short-term teriparatide administration on healing of autologous bone graft in mandibular critical-size defects. SUBJECTS AND METHODS:A 5-mm mandibular bone defect was created and iliac bone graft was harvested in 135 rats. Rats were randomly divided into 3 groups of negative control (NC), control (C), and study (S). In groups S and C, iliac graft was placed in defect and 2 μg/kg/day teriparatide or saline, respectively, was administered for 20 days. In group NC, iliac graft was not transferred to the defect and saline was injected for 20 days. Twenty, 40, and 60 days after surgery, 15 rats in each group were euthanized and the healing process was histologically evaluated and scored using a grading system (1-6). RESULTS:In group NC, defects did not heal or were predominantly filled with fibrous tissue. At day 20, bone defects in both C and S groups contained a large area of graft particles, numerous collagen fibers and some areas of new trabeculae. At the day 40, defects in group S showed a larger bone graft area, more new bone formation, smaller connective tissue area, and a higher healing score compared to group C (P < 0.05). At day 60, most of the defect in group S was filled with graft particles and mature bone while in group C, new trabecular bone formation was still underway (P < 0.05). CONCLUSION:Teriparatide therapy improves healing of bone defects reconstructed with autograft by reducing bone graft resorption and enhancing new bone formation and maturation.
Drug-loading three-dimensional scaffolds based on hydroxyapatite-sodium alginate for bone regeneration.
Liang Tingting,Wu Jingwen,Li Fuyao,Huang Zhu,Pi Yixing,Miao Guohou,Ren Wen,Liu Tiantao,Jiang Qianzhou,Guo Lvhua
Journal of biomedical materials research. Part A
Bone tissue engineering is a promising approach for tackling clinical challenges. Osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds are employed in bone tissue engineering. However, scaffold materials remain limited due to their source, low biocompatibility, and so on. In this study, a composite hydrogel scaffold composed of hydroxyapatite (HA) and sodium alginate (SA) was manufactured using three-dimensional printing. Naringin (NG) and calcitonin-gene-related peptide (CGRP) were used as osteogenic factors in the fabrication of drug-loaded scaffolds. Investigation using animal experiments, as well as scanning electron microscopy, cell counting kit-8 testing, alkaline phosphatase staining, and alizarin red-D staining of bone marrow mesenchymal stem cell culture showed that the three scaffolds displayed similar physicochemical properties and that the HA/SA/NG and HA/SA/CGRP scaffolds displayed better osteogenesis than that of the HA/SA scaffold. Thus, the HA/SA scaffold could be a biocompatible material with potential applications in bone regeneration. Meanwhile, NG and CGRP doping could result in better and more positive proliferation and differentiation.
Maxillary sinus augmentation using a peptide-modified graft material in three mixtures: a prospective human case series of histologic and histomorphometric results.
Gelbart Michael,Friedman Robert,Burlui Vasile,Rohrer Michael,Atkinson Brent
This prospective study assessed vital bone quality and quantity after grafting maxillary sinuses with anorganic bone mineral containing a cell binding peptide (ABM/P-15) in combination with DFDBA (Mix I), hydrogel/ABM/P-15 (Mix II), or PRP (Mix III). Fifteen maxillary sinuses in 12 patients were grafted with the ABM/P-15 mixtures and after 4 to 5 months, cores were taken and analyzed histologically and histomorphometrically. Fifty-nine screw-type implants were placed. Mixes containing a spacer material (Mix I and II) produced greater average vital bone, more mature bone, and more interconnected bone bridges from the cortical (oral) end to the most apical portion, compared to a mix that lacked a spacer material (Mix III). None of the 59 implants failed with any graft material up to 5 months following implant placement. The study demonstrates that new trabecular bone is formed after grafting ABM/P-15 in the sinus floor; that more vital bone is formed when ABM/P-15 is mixed with a spacer material than without; and that implants can be successfully placed.
Alginate/Poly(γ-glutamic Acid) Base Biocompatible Gel for Bone Tissue Engineering.
Chan Wing P,Kung Fu-Chen,Kuo Yu-Lin,Yang Ming-Chen,Lai Wen-Fu Thomas
BioMed research international
A technique for synthesizing biocompatible hydrogels by cross-linking calcium-form poly(γ-glutamic acid), alginate sodium, and Pluronic F-127 was created, in which alginate can be cross-linked by Ca(2+) from Ca-γ-PGA directly and γ-PGA molecules introduced into the alginate matrix to provide pH sensitivity and hemostasis. Mechanical properties, swelling behavior, and blood compatibility were investigated for each hydrogel compared with alginate and for γ-PGA hydrogel with the sodium form only. Adding F-127 improves mechanical properties efficiently and influences the temperature-sensitive swelling of the hydrogels but also has a minor effect on pH-sensitive swelling and promotes anticoagulation. MG-63 cells were used to test biocompatibility. Gelation occurred gradually through change in the elastic modulus as the release of calcium ions increased over time and caused ionic cross-linking, which promotes the elasticity of gel. In addition, the growth of MG-63 cells in the gel reflected nontoxicity. These results showed that this biocompatible scaffold has potential for application in bone materials.
Evaluation of parathyroid hormone bound to a synthetic matrix for guided bone regeneration around dental implants: a histomorphometric study in dogs.
Valderrama Pilar,Jung Ronald E,Thoma Daniel S,Jones Archie A,Cochran David L
Journal of periodontology
BACKGROUND:A polyethylene glycol (PEG)-based hydrogel matrix covalently bound to a 35-amino acid peptide of parathyroid hormone cystein-PTH 1-34 (cys-PTH 1-34) was shown to enhance bone regeneration around implants. The aim of this study is to test if the addition of an integrin-receptor-binding arginine-glycine-aspartic acid (RGD)-containing peptide at early healing time points improves the performance of the PEG matrix supplemented with cys-PTH 1-34 (PTH) when applied in acute defects around implants at early healing time points (2 and 4 weeks). METHODS:Six dogs received 48 implants. Each side of the mandible was randomly assigned for implantation at day 0 or 2 weeks. A circumferential critical-size defect was created at each site before implantation. Sites were randomly assigned to one of four groups: 1) PEG alone (PEG group), 2) PEG plus RGD (PEG/RGD group), 3) PEG plus PTH (PEG/PTH group), and 4) PEG plus RGD plus PTH (PEG/RGD/PTH group). Dogs were sacrificed 2 weeks after the second surgery, and specimens were obtained for histologic analysis. For the statistical analysis, mixed linear regression with repeated measurements was used, and a Dunnett-Hsu adjustment was made for multiple comparisons. RESULTS:At 2 weeks, the percentages of new bone formation within the defect were 12.43% for the PEG group, 15.95% for the PEG/RGD group, 15.32% for the PEG/PTH group, and 16.60% for the PEG/RGD/PTH group. At 4 weeks, the percentages of new bone formation within the defect were 30.01% for the PEG group, 27.90% for the PEG/RGD group, 29.89% for the PEG/PTH group, and 27.58% for the PEG/RGD/PTH group. A marginally significant difference (PEG/RGD/PTH group versus PEG group; P = 0.055) was found at 2 weeks but not at 4 weeks. The highest percentage of bone-to-implant contact (BIC) in the defect site at 2 weeks was observed for the PEG/RGD group (8.57%). The BIC after 4 weeks of healing ranged from 11.54% (PEG/RGD/PTH group) to 16.61% (PEG group). No statistically significant differences were observed in BIC. CONCLUSIONS:The effect of binding PTH covalently to a synthetic, RGD-modified PEG hydrogel marginally significantly improved bone formation at 2 weeks of healing compared to the use of PEG alone. Bone regeneration within the defects increased in all groups at week 4 of healing without statistically significant differences.
Composition of elastin like polypeptide-collagen composite scaffold influences in vitro osteogenic activity of human adipose derived stem cells.
Gurumurthy Bhuvaneswari,Bierdeman Patrick C,Janorkar Amol V
Dental materials : official publication of the Academy of Dental Materials
OBJECTIVE:Collagen-based scaffolds for guided bone regeneration (GBR) are continuously improved to overcome the mechanical weaknesses of collagen. We have previously demonstrated superior mechanical characteristics of the elastin-like polypeptide (ELP) reinforced collagen composites. The objectives of this research were to evaluate the efficacy of ELP-collagen composites to culture human adipose-derived stem cells (hASCs) and allow them to undergo osteogenic differentiation. We hypothesized that hASCs would show a superior osteogenic differentiation in stiffer ELP-collagen composites compared to the neat collagen hydrogels. METHODS:Composite specimens were made by varying ELP (0-18mg/mL) and collagen (2-6mg/mL) in a 3:1 ratio. Tensile strength, elastic modulus, and toughness were determined by uniaxial tensile testing. hASCs cultured within the composites were characterized by biochemical assays to measure cell viability, protein content, and osteogenic differentiation (alkaline phosphatase activity, osteocalcin, and Alizarin red staining). Scanning electron microscopy and energy dispersive spectroscopy were used for morphological characterization of composites. RESULTS:All composites were suitable for hASCs culture with viable cells over the 22-day culture period. The ELP-collagen composite with 18mg/mL of ELP and 6mg/mL of collagen had greater tensile strength and elastic modulus combined with higher osteogenic activity in terms of differentiation and subsequent mineralization over a period of 3 weeks compared to other compositions. The extra-cellular matrix deposits composed of calcium and phosphorous were specifically seen in the 18:6mg/mL ELP-collagen composite. SIGNIFICANCE:The success of the 18:6mg/mL ELP-collagen composite to achieve long-term, 3-dimensional culture and osteogenic differentiation indicates its potential as a GBR scaffold.
The use of injectable, thermosensitive poly(organophosphazene)-RGD conjugates for the enhancement of mesenchymal stem cell osteogenic differentiation.
Chun Changju,Lim Hye Jin,Hong Ki-Yun,Park Keun-Hong,Song Soo-Chang
An injectable and thermosensitive poly(organophosphazene)-RGD conjugate to enhance functionality was synthesized by a covalent amide linkage between a cell adhesion peptide, GRGDS and carboxylic acid-terminated poly(organophosphazene). The aqueous solutions of synthesized poly(organophosphazene)-GRGDS conjugates existed in an injectable fluid state at room temperature and immediately formed a hydrogel at body temperature. The rabbit mesenchymal stem cells (rMSCs) on the polymer-GRGDS conjugate (conjugate 1-2, 0.05 mol fraction as GRGDS) hydrogel constructs using an injection method into a nude mouse were proved to express markers at mRNA level for all stages towards osteogenesis and mainly a sharp increase of osteocalcin (OCN, a typical late osteogenic differentiation marker) levels at 4th week post-induction indicated that the maturation process has started within this period. By histological and immunohistochemical evaluations, significantly high mineralization level by calcium contents was detected qualitatively and collagen type I (Col I), a major characteristic marker protein, was mainly and highly expressed by the rMSCs cultivated in the polymer-GRGDS conjugate hydrogel constructs formed into the nude mouse. The results suggest that the poly(organophosphazene)-GRGDS conjugate to enhance biofunctionality hold a promise for cell delivery material to induce osteogenic differentiation of MSC for enhancing ectopic bone formation.
Biphasic peptide amphiphile nanomatrix embedded with hydroxyapatite nanoparticles for stimulated osteoinductive response.
Anderson Joel M,Patterson Jessica L,Vines Jeremy B,Javed Amjad,Gilbert Shawn R,Jun Ho-Wook
Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprising self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e., RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration.
Peptide-induced de novo bone formation after tooth extraction prevents alveolar bone loss in a murine tooth extraction model.
Arai Yuki,Aoki Kazuhiro,Shimizu Yasuhiro,Tabata Yasuhiko,Ono Takashi,Murali Ramachandran,Mise-Omata Setsuko,Wakabayashi Noriyuki
European journal of pharmacology
Tooth extraction causes bone resorption of the alveolar bone volume. Although recombinant human bone morphogenetic protein 2 (rhBMP-2) markedly promotes de novo bone formation after tooth extraction, the application of high-dose rhBMP-2 may induce side effects, such as swelling, seroma, and an increased cancer risk. Therefore, reduction of the necessary dose of rhBMP-2 which can still obtain sufficient bone mass is necessary by developing a new osteogenic reagent. Recently, we showed that the systemic administration of OP3-4 peptide, which was originally designed as a bone resorption inhibitor, had osteogenic ability both in vitro and in vivo. This study evaluated the ability of the local application of OP3-4 peptide to promote bone formation in a murine tooth extraction model with a very low-dose of BMP. The mandibular incisor was extracted from 10-week-old C57BL6/J male mice and a gelatin hydrogel containing rhBMP-2 with or without OP3-4 peptide (BMP/OP3-4) was applied to the socket of the incisor. Bone formation inside the socket was examined radiologically and histologically at 21 days after the extraction. The BMP/OP3-4-group showed significant bone formation inside the mandibular extraction socket compared to the gelatin-hydrogel-carrier-control group or rhBMP-2-applied group. The BMP/OP3-4-applied mice showed a lower reduction of alveolar bone and fewer osteoclast numbers, suggesting that the newly formed bone inside the socket may prevent resorption of the cortical bone around the extraction socket. Our data revealed that OP3-4 peptide promotes BMP-mediated bone formation inside the extraction socket of mandibular bone, resulting in preservation from the loss of alveolar bone.
Feasibility and effects of a self-assembling peptide as a scaffold in bone healing: An in vivo study in rabbit lumbar posterolateral fusion and tibial intramedullary models.
Ando Kei,Imagama Shiro,Kobayashi Kazuyoshi,Ito Kenyu,Tsushima Mikito,Morozumi Masayoshi,Tanaka Satoshi,Machino Masaaki,Ota Kyotaro,Nishida Koji,Nishida Yoshihiro,Ishiguro Naoki
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Spinal fusion and bone defects after injuries, removal of bone tumors, and infections require repair by implantation. In this study, we show self-assembling peptide (SPG-178) hydrogel-induced bone healing in vivo. Posterolateral lumbar fusion and tibial intramedullary models of rabbits were prepared. In the tibia model, micro-CT analysis revealed a significantly higher degree of newly formed bone matrix in the SPG-178 group compared to the other groups. SEM/3D micrographs showed that the cavity filled with SPG-178 had collagen fibers attached to host bone. After 28 days, samples from the SPG-178 group showed significant repair of the defect. In the posterolateral lumbar fusion models, micro-CT showed a tendency for a higher degree of newly formed bone matrix in the SPG-178 group compared to the β-TCP and bone chips only groups. Von Kossa staining showed marked new bone formation attached to the lamina that was most prominent at the implanted SPG-178 composite margin. SPG-178 is a material that is likely to be used in clinical applications because it has several benefits. These include its favorable bone conduction properties, its ability to act as a support for various cells and growth factors, its lack of infection risk compared with materials of animal origin such as ECM, and the ease with which it can be used to fill defects with complex shapes and be combined with a wide range of other materials. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3285-3293, 2018.
Influence of discrete and continuous culture conditions on human mesenchymal stem cell lineage choice in RGD concentration gradient hydrogels.
Smith Callahan Laura A,Policastro Gina M,Bernard Sharon L,Childers Erin P,Boettcher Ronna,Becker Matthew L
Stem cells have shown lineage-specific differentiation when cultured on substrates possessing signaling groups derived from the native tissue. A distinct determinant in this process is the concentration of the signaling motif. While several groups have been working actively to determine the specific factors, concentrations, and mechanisms governing the differentiation process, many have been turning to combinatorial and gradient approaches in attempts to optimize the multiple chemical and physical parameters needed for the next advance. However, there has not been a direct comparison between the cellular behavior and differentiation of human mesenchymal stem cells cultured in gradient and discrete substrates, which quantitates the effect of differences caused by cell-produced, soluble factors due to design differences between the culture systems. In this study, the differentiation of human mesenchymal stem cells in continuous and discrete polyethylene glycol dimethacrylate (PEGDM) hydrogels containing an RGD concentration gradient from 0 to 14 mM were examined to study the effects of the different culture conditions on stem-cell behavior. Culture condition was found to affect every osteogenic (alkaline phosphatase, Runx 2, type 1 collagen, bone sailoprotein, and calcium content) and adipogenic marker (oil red and peroxisome proliferator-activated receptor gamma) examined regardless of RGD concentration. Only in the continuous gradient culture did RGD concentration affect human mesenchymal stem-cell lineage commitment with low RGD concentrations expressing higher osteogenic differentiation than high RGD concentrations. Conversely, high RGD concentrations expressed higher adipogenic differentiation than low RGD concentrations. Cytoskeletal actin organization was only affected by culture condition at low RGD concentrations, indicating that it played a limited role in the differences in lineage commitment observed. Therefore, the role of discrete versus gradient strategies in high-throughput experimentation needs to be considered when designing experiments as we show that the respective strategies alter cellular outcomes even though base scaffolds have similar material and chemical properties.
Controlled release of simvastatin from in situ forming hydrogel triggers bone formation in MC3T3-E1 cells.
Park Yoon Shin,David Allan E,Park Kyung Min,Lin Chia-Ying,Than Khoi D,Lee Kyuri,Park Jun Beom,Jo Inho,Park Ki Dong,Yang Victor C
The AAPS journal
Simvastatin (SIM), a drug commonly administered for the treatment of hypercholesterolemia, has been recently reported to induce bone regeneration/formation. In this study, we investigated the properties of hydrogel composed of gelatin-poly(ethylene glycol)-tyramine (GPT) as an efficient SIM delivery vehicle that can trigger osteogenic differentiation. Sustained delivery of SIM was achieved through its encapsulation in an injectable, biodegradable GPT-hydrogel. Cross-linking of the gelatin-based GPT-hydrogel was induced by the reaction of horse radish peroxidase and H(2)O(2). GPT-hydrogels of three different matrix stiffness, 1,800 (GPT-hydrogel1), 5,800 (GPT-hydrogel2), and 8,400 Pa (GPT-hydrogel3) were used. The gelation/degradation time and SIM release profiles of hydrogels loaded with two different concentrations of SIM, 1 and 3 mg/ml, were also evaluated. Maximum swelling times of GPT-hydrogel1, GPT-hydrogel2, and GPT-hydrogel3 were observed to be 6, 12, and 20 days, respectively. All GPT-hydrogels showed complete degradation within 55 days. The in vitro SIM release profiles, investigated in PBS buffer (pH 7.4) at 37°C, exhibited typical biphasic release patterns with the initial burst being more rapid with GPT-hydrogel1 compared with GPT-hydrogel3. Substantial increase in matrix metalloproteinase-13, osteocalcin expression levels, and mineralization were seen in osteogenic differentiation system using MC3T3-E1 cells cultured with GPT-hydrogels loaded with SIM in a dose-dependent manner. This study demonstrated that controlled release of SIM from a biodegradable, injectable GPT-hydrogel had a promising role for long-term treatment of chronic degenerative diseases such as disc degenerative disease.
Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model.
Neuerburg Carl,Mittlmeier Lena M,Keppler Alexander M,Westphal Ines,Glass Änne,Saller Maximilian M,Herlyn Philipp K E,Richter Heiko,Böcker Wolfgang,Schieker Matthias,Aszodi Attila,Fischer Dagmar-C
Journal of orthopaedic surgery and research
BACKGROUND:Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible. METHODS:In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 μl saline, 100 μl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 μl hydrogel loaded with 1 μg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination. RESULTS:Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples. CONCLUSIONS:In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.
[Biocompatibility and osteoinductive properties of collagen and fibronectin hydrogel impregnated with rhBMP-2].
Vasilyev A V,Kuznetsova V S,Galitsyna E V,Bukharova T B,Osidak E O,Fatkhudinova N L,Leonov G E,Babichenko I I,Domogatsky S P,Goldstein D V,Kulakov A A
The study aimed to demonstrate the biocompatibility and osteoinductive properties of a hydrogel based on highly purified collagen and fibronectin impregnated with rhBMP-2. In vitro and in vivo experiments have shown that the minimum effective dosage of rhBMP-2 is 10 μg/ml. The cytocompatibility of the collagen-fibronectin gel was determined using MTT test and staining with PKH-26. There was no inflammation reaction when the material was subcutaneously implanted in rats (n=30) in vivo. The collagen-fibronectin hydrogel containing 10 μg/ml rhBMP-2 showed high osteogenic properties. By the end of 28 days 8±4% of its volume was replaced by newly formed bone tissue in case of subcutaneous implantation, 17±10% in intramuscular implantation and 26±11% in intraosseous implantation in the calvarial critical-size. The optimal combination of biocompatible and osteogenic properties of collagen-fibronectin hydrogel impregnated with BMP-2 allows us to consider it as a promising basis for creating the new generation of osteoplastic materials for dentistry.
[ study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustained-release system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells].
Chai Le,Quan Renfu,Hu Jintao,Huang Xiaolong,Lü Jianlan,Zhang Can,Qiu Rui,Cai Bingbing
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery
Objective:To construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO ) bio porous ceramic foam, co-culture , and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs. Methods:BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and sustained release rate of the microspheres were tested. HA/ZrO bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy. Results:BMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the co-culture system ( <0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well. Conclusion:iPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
Time-responsive osteogenic niche of stem cells: A sequentially triggered, dual-peptide loaded, alginate hybrid system for promoting cell activity and osteo-differentiation.
Luo Zuyuan,Zhang Siqi,Pan Jijia,Shi Rui,Liu Hao,Lyu Yalin,Han Xiao,Li Yan,Yang Yue,Xu Zhixiu,Sui Yi,Luo En,Zhang Yuanyuan,Wei Shicheng
The efficacy of stem cell-based bone tissue engineering has been hampered by cell death and limited fate control. A smart cell culture system with the capability of sequentially delivering multiple factors in specific growth stages, like the mechanism of the natural extracellular matrix modulating tissue formation, is attractive for enhancing cell activity and controlling cell fate. Here, a bone forming peptide-1 (BFP-1)-laden mesoporous silica nanoparticles (pep@MSNs) incorporated adhesion peptide, containing the arginine-glycine-aspartic acid (RGD) domain, modified alginate hydrogel (RA) system (pep@MSNs-RA) was developed to promote the activity and stimulate osteo-differentiation of human mesenchymal stem cells (hMSCs) in sequence. The survivability and proliferation of hMSCs were enhanced in the adhesion peptide modified hydrogel. Next, BFP-1 released from pep@MSNs induced hMSCs osteo-differentiation after the proliferation stage. Moreover, BFP-1 near the cells was self-captured by the additional cell-peptide cross-linked networks formed by the ligands (RGD) binding to receptors on the cell surface, leading to long-term sustained osteo-stimulation of hMSCs. The results suggest that independent and sequential stimulation in proliferation and osteo-differentiation stages could synergistically enhance the survivability, expansion, and osteogenesis of hMSCs, as compared to stimulating alone or simultaneously. Overall, this study provided a new and valid strategy for stem cell expansion and osteo-differentiation in 2D or 3D culture systems, possessing potential applications in 3D bio-printing and tissue regeneration.
Immobilization of BMP-2-derived peptides on 3D-printed porous scaffolds for enhanced osteogenesis.
Zhang Xiashiyao,Lou Qi,Wang Lili,Min Shan,Zhao Meng,Quan Changyun
Biomedical materials (Bristol, England)
Three-dimensional (3D) printing technologies open up new perspectives for customizing the external shape and internal architecture of bone scaffolds. In this study, an oligopeptide (SSVPT, Ser-Ser-Val-Pro-Thr) derived from bone morphogenetic protein 2 was conjugated with a dopamine coating on a 3D-printed poly(lactic acid) (PLA) scaffold to enhance osteogenesis. Cell experiments in vitro showed that the scaffold was highly osteoconductive to the adhesion and proliferation of rat marrow mesenchymal stem cells (MSCs). In addition, RT-PCR analysis showed that the scaffold was able to promote the expression of osteogenesis-related genes, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and osteopontin (OPN). Images of the micro-CT 3D reconstruction from the rat cranial bone defect model showed that bone regeneration patterns occurred from one side edge towards the center of the area implanted with the prepared biomimetic peptide hydrogels, demonstrating significantly accelerated bone regeneration. This work will provide a basis to explore the application potential of bioactive scaffolds further.
Silk fibroin/nanohydroxyapatite hydrogels for promoted bioactivity and osteoblastic proliferation and differentiation of human bone marrow stromal cells.
Ribeiro Marta,Fernandes Maria H,Beppu Marisa M,Monteiro Fernando J,Ferraz Maria P
Materials science & engineering. C, Materials for biological applications
Silk fibroin (SF) is a natural, biocompatible, and biodegradable polymer having a great potential for the successful regeneration of damaged bone tissue. In the present work, nanohydroxyapatite (nanoHA) was incorporated into SF polymer to form a bioactive composite hydrogel for applications as bone implants. The degradation and bioactive properties of SF/nanoHA composite hydrogels were evaluated. Additionally, biological investigations of human bone marrow stromal cells (hBMSCs) viability, proliferation and differentiation to the osteoblastic phenotype were conducted. The incorporation of nanoHA in SF polymer matrices improved the bioactivity of the hydrogels. The biological results highlighted that the SF/nanoHA composite hydrogels are suitable for hBMSCs attachment and proliferation, while a test for alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) expression suggested osteoblast differentiation. Additionally, a cell staining method for ALP allowed to observe cell infiltration with active production of ALP by the infiltrated cells, paving the way to use the proposed composite hydrogel for bone tissue regeneration.
Efficient in vivo bone formation by BMP-2 engineered human mesenchymal stem cells encapsulated in a projection stereolithographically fabricated hydrogel scaffold.
Lin Hang,Tang Ying,Lozito Thomas P,Oyster Nicholas,Wang Bing,Tuan Rocky S
Stem cell research & therapy
BACKGROUND:Stem cell-based bone tissue engineering shows promise for bone repair but faces some challenges, such as insufficient osteogenesis and limited architecture flexibility of the cell-delivery scaffold. METHODS:In this study, we first used lentiviral constructs to transduce ex vivo human bone marrow-derived stem cells with human bone morphogenetic protein-2 (BMP-2) gene (BMP-hBMSCs). We then introduced these cells into a hydrogel scaffold using an advanced visible light-based projection stereolithography (VL-PSL) technology, which is compatible with concomitant cell encapsulation and amenable to computer-aided architectural design, to fabricate scaffolds fitting local physical and structural variations in different bones and defects. RESULTS:The results showed that the BMP-hBMSCs encapsulated within the scaffolds had high viability with sustained BMP-2 gene expression and differentiated toward an osteogenic lineage without the supplement of additional BMP-2 protein. In vivo bone formation efficacy was further assessed using an intramuscular implantation model in severe combined immunodeficiency (SCID) mice. Microcomputed tomography (micro-CT) imaging indicated rapid bone formation by the BMP-hBMSC-laden constructs as early as 14 days post-implantation. Histological examination revealed a mature trabecular bone structure with considerable vascularization. Through tracking of the implanted cells, we also found that BMP-hBMSC were directly involved in the new bone formation. CONCLUSIONS:The robust, self-driven osteogenic capability and computer-designed architecture of the construct developed in this study should have potential applications for customized clinical repair of large bone defects or non-unions.
Enhanced healing of surgical periodontal defects in rats following application of a self-assembling peptide nanofibre hydrogel.
Takeuchi Takahiro,Bizenjima Takahiro,Ishii Yoshihito,Imamura Kentaro,Suzuki Eiichi,Seshima Fumi,Saito Atsushi
Journal of clinical periodontology
AIM:The aim of this study was to investigate the effects of a self-assembling peptide (SAP) nanofibre hydrogel on healing of surgical periodontal defects in rats. MATERIALS AND METHODS:In vitro interactions between rat periodontal ligament (PDL) cells and SAP hydrogel (2.5% RADA16) were assessed by cell proliferation assays. In vivo, maxillary first molars of 45 Wistar rats were extracted and after healing, bilateral periodontal defects were surgically created mesially in second molars. Defects were treated with RADA16, Matrigel, or left unfilled. After 2 and 4 weeks, defect healing was evaluated by microcomputed tomography, histological and immunohistochemical analyses. RESULTS:Periodontal ligament cells grown on RADA16 showed an gradual increase in proliferation up to 72 h. At 4 weeks post surgery, the bone volume fraction and trabecular thickness of defect areas in the RADA16 group were significantly greater than those in other groups. Histologically, enhanced new bone formation was observed in the RADA16 group. At 4 weeks, PDL-like collagen bundles ran oblique to the root surface in the RADA16 group. Expression levels of PCNA-positive cells, vascular endothelial growth factor and osteopontin in the RADA16 group were significantly greater than those in other groups. CONCLUSIONS:Within the limitations of the study, application of the SAP hydrogel promoted healing of surgical periodontal defects by enhancing cell recruitment and possibly angiogenesis.
Osteogenic efficacy of BMP-2 mixed with hydrogel and bone substitute in peri-implant dehiscence defects in dogs: 16 weeks of healing.
Cha Jae-Kook,Jung Ui-Won,Thoma Daniel S,Hämmerle Christoph H F,Jung Ronald E
Clinical oral implants research
OBJECTIVES:The objective of this study was to determine the effect of bone morphogenetic protein-2 (BMP-2) mixed with either polyethylene glycol hydrogel or synthetic bone substitute (SBS) on new bone formation in peri-implant dehiscence defects after 16 weeks of healing. MATERIALS AND METHODS:A guided bone regeneration procedure was performed in box-type peri-implant defects that were surgically prepared in six beagle dogs. The following four experimental groups were used (i) control (no graft), (ii) SBS+hydrogel, (iii) SBS+BMP-2/hydrogel and (iv) BMP-2/SBS+hydrogel. Volumetric analysis using micro-computed tomography and histomorphometric analysis was performed at 16 weeks post-operatively. RESULTS:The amount of new bone and the total augmented volume did not differ significantly between both BMP-treated groups and the SBS+hydrogel group (p > .05). Likewise, no histometric differences were observed in the values of new bone area and bone-to-implant contact ratio among the three augmentation groups (new bone area: 0.06 ± 0.08, 0.19 ± 0.20, 0.48 ± 0.37 and 0.56 ± 0.60 mm [mean ± standard deviation] in groups 1-4, respectively; bone-to-implant contact: 9.44 ± 11.51%, 19.91 ± 15.19%, 46.31 ± 29.82% and 42.58 ± 26.27% in groups 1-4, respectively). CONCLUSION:The osteogenic efficacy of BMP-2 on the regeneration of peri-implant bone defects was not detectable after 16 weeks regardless of the carrier materials.
Photocrosslinkable chitosan hydrogels functionalized with the RGD peptide and phosphoserine to enhance osteogenesis.
Kim Soyon,Cui Zhong-Kai,Fan Jiabing,Fartash Armita,Aghaloo Tara L,Lee Min
Journal of materials chemistry. B
Hydrogels derived from naturally occurring polymers are attractive matrix for tissue engineering. Here, we report a biofunctional hydrogel for specific use in bone regeneration by introducing Arg-Gly-Asp (RGD)-containing cell adhesive motifs and phosphorylated serine residues, which are prevalent in native bone extracellular matrix and known to promote osteogenesis by enhancing cell-matrix interactions and hydroxyapatite nucleation, into photopolymerizable methacrylated glycol chitosan (MeGC). Incorporation of phosphoserine into MeGC hydrogels increased the ability of the hydrogels to nucleate mineral on their surfaces. RGD incorporation enhanced cell-matrix interactions by supporting attachment, spreading, and proliferation of bone marrow stromal cells (BMSCs) encapsulated in the hydrogels. Moreover, co-modification of MeGC hydrogels with RGD and phosphoserine synergistically increased osteogenic differentiation of encapsulated BMSCs . The bone healing capacity of the modified hydrogels was further confirmed in a mouse calvarial defect model. These findings suggest a promising hydrogel platform with a specific microenvironment tailored to promote osteogenesis for clinical bone repair.
Photo-crosslinkable, bone marrow-derived mesenchymal stem cells-encapsulating hydrogel based on collagen for osteogenic differentiation.
Zhang Tingting,Chen Hong,Zhang Yajie,Zan Yue,Ni Tianyu,Liu Min,Pei Renjun
Colloids and surfaces. B, Biointerfaces
Many patients suffer from bone injury and self-regeneration is not effective. Developing new strategies for effective bone injury repair is highly desired. Herein, collagen, an important component of the extracellular matrix, was modified with glycidyl methacrylate. The water solubility and photochemical cross-linking ability of the resulting collagen derivative was then improved. Thereafter, BMSC-laden hydrogel was fabricated using collagen modified with glycidyl methacrylate and hyaluronic acid modified with methacrylic anhydride under UV light in the presence of I 2959. The physicochemical properties were characterized suggesting that the hydrogel had great potential for enhancing cell adhesion and proliferation. Furthermore, without adding the bone morphogenetic protein-2, the collagen also promoted osteogenic differentiation of BMSCs within the hydrogel. Altogether, this hydrogel system provides a general strategy to fabricate cell-encapsulating hydrogel based on collagen and could be used as 3D scaffold for bone injury repair.
Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects.
Kim Sungwoo,Bedigrew Katherine,Guda Teja,Maloney William J,Park Sangwon,Wenke Joseph C,Yang Yunzhi Peter
The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing a CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100 and 500ngml(-1)) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2 and 5μg) on bone formation was evaluated using rat critical size segmental bone defects for 4weeks. Fourier transform infrared spectroscopy spectra and scanning electron microscopy images showed chemical and structural changes by the addition of fibrinogen into the chitosan-lactide copolymer. The incorporation of fibrinogen molecules significantly increased the compressive modulus of the hydrogels. The in vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiography, microcomputed tomography and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.
Design of hydrogels to stabilize and enhance bone morphogenetic protein activity by heparin mimetics.
Kim Soyon,Cui Zhong-Kai,Kim Paul Jay,Jung Lawrence Young,Lee Min
Although bone morphogenetic protein-2 (BMP-2) is known to be the most potent stimulator available for bone formation, a major barrier to widespread clinical use is its inherent instability and absence of an adequate delivery system. Heparin is being widely used in controlled release systems due to its strong binding ability and protective effect for many growth factor proteins. In this work, we developed a hydrogel surface that can mimic heparin to stabilize BMP-2 and to enhance osteogenesis by introducing heparin-mimicking sulfonated molecules such as poly-vinylsulfonic acid (PVSA) or poly-4-styrenesulfonic acid (PSS), into photo-crosslinkable hydrogel. Bioactivity of BMP-2 was well preserved in the presence of polysulfonates during exposure to various therapeutically relevant stressors. The heparin-mimicking sulfonated hydrogels were effective to bind BMP-2 compared to unmodified MeGC hydrogel and significantly enhanced osteogenic differentiation of encapsulated bone marrow stromal cells (BMSCs) without the addition of exogenous BMP-2. The sulfonated hydrogels were effective in delivering exogenous BMP-2 with reduced initial burst and increased BMSCs osteogenesis induced by BMP-2. These findings suggest a novel hydrogel platform for sequestering and stabilizing BMP-2 to enhance osteoinductive activity in bone tissue engineering. STATEMENT OF SIGNIFICANCE:Although bone morphogenetic protein-2 (BMP-2) is believed to be the most potent cytokine for bone regeneration, its clinical applications require supraphysiological BMP dosage due to its intrinsic instability and fast enzymatic degradation, leading to worrisome side effects. This study demonstrates a novel hydrogel platform that mimics a natural protector of BMPs, heparin, to sequester and stabilize BMP-2 for increased osteoinductive signaling. This study will achieve the stabilization of BMPs with prolonged bioactivity by a synthetic heparin mimic that has not been examined previously. Moreover, the heparin mimetic hydrogel surface can augment endogenous BMP activity by sequestering and localizing the cell-produced BMPs. The additional knowledge gained from this study may suggest basis for future development of material-based therapeutics for tissue engineering.
Development of CaCO microsphere-based composite hydrogel for dual delivery of growth factor and Ca to enhance bone regeneration.
Gong Yihong,Zhang Yiling,Cao Zhinan,Ye Feng,Lin Zefeng,Li Yan
Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO3 microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO3 microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO3 had a microsphere morphology of ∼1 μm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO3 microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO3 significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.
Bone induction through controlled release of novel BMP-2-related peptide from PTMC₁₁-F127-PTMC₁₁ hydrogels.
Tang Shuo,Zhao Jingjing,Xu Shuyun,Li Jingfeng,Teng Yu,Quan Daping,Guo Xiaodong
Biomedical materials (Bristol, England)
Bone morphogenetic protein 2 (BMP-2) is the most powerful osteogenic factor; its effectiveness in enhancing osteoblastic activation has been confirmed both in vitro and in vivo. We developed a novel peptide (designated P24) derived from the 'knuckle' epitope of BMP-2 and found it also had osteogenic bioactivity to some extent. The main objective of this study was to develop a controlled release system based on poly(trimethylene carbonate)-F127-poly(trimethylene carbonate) (PTMC₁₁-F127-PTMC₁₁) hydrogels for the P24 peptide, to promote bone formation. By varying the copolymer concentrations, we demonstrated that P24/PTMC₁₁-F127-PTMC₁₁ hydrogels were an efficient system for the sustained release of P24 over 21-35 days. The P24-loaded hydrogels elevated alkaline phosphatase activity and promoted the expression of osteocalcin mRNA in bone marrow stromal cells (BMSCs) in vitro. Radiographic and histological examination showed that P24-loaded hydrogels could induce more effective ectopic bone formation in vivo than P24-free hydrogels. These results indicate that the PTMC₁₁-F127-PTMC₁₁ hydrogel is a suitable carrier for the controlled release of P24, and is a promising injectable biomaterial for the induction of bone regeneration.
Sustained Release of Two Bioactive Factors from Supramolecular Hydrogel Promotes Periodontal Bone Regeneration.
Tan Jiali,Zhang Mei,Hai Zijuan,Wu Chengfan,Lin Jiong,Kuang Wen,Tang Hang,Huang Yulei,Chen Xiaodan,Liang Gaolin
Intact and stable bone reconstruction is ideal for the treatment of periodontal bone destruction but remains challenging. In research, biomaterials are used to encapsulate stem cells or bioactive factors for periodontal bone regeneration, but, to the best of our knowledge, using a supramolecular hydrogel to encapsulate bioactive factors for their sustained release in bone defect areas to promote periodontal bone regeneration has not been reported. Herein, we used a well-studied hydrogelator, NapFFY, to coassemble with SDF-1 and BMP-2 to prepare a supramolecular hydrogel, SDF-1/BMP-2/NapFFY. In vitro and in vivo results indicated that these two bioactive factors were ideally, synchronously, and continuously released from the hydrogel to effectively promote the regeneration and reconstruction of periodontal bone tissues. Specifically, after the bone defect areas were treated with our SDF-1/BMP-2/NapFFY hydrogel for 8 weeks using maxillary critical-sized periodontal bone defect model rats, a superior bone regeneration rate of 56.7% bone volume fraction was achieved in these rats. We anticipate that our SDF-1/BMP-2/NapFFY hydrogel could replace bone transplantation in the clinic for the repair of periodontal bone defects and periodontally accelerated osteogenic orthodontics in the near future.
Thiol-ene Hydrogels for Local Delivery of PTH for Bone Regeneration in Critical Size defects.
Wojda Samantha J,Marozas Ian A,Anseth Kristi S,Yaszemski Michael J,Donahue Seth W
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Neither allograft nor commercially available bone graft substitutes provide the same quality of bone healing as autograft. Incorporation of bioactive molecules like parathyroid hormone (PTH) within bone graft substitute materials may provide similar, if not better treatment options to grafting. The goal of this work was to develop a biomaterial system for the local delivery of PTH to large bone defects for promoting bone regeneration. PTH was loaded in a thiol-ene hydrogel at several concentrations and polymerized in and around an osteoconductive poly(propylene fumarate) (PPF) scaffold. PTH was shown to be bioactive when released from the hydrogel for up to 21 days. Eighty percent of the PTH was released by day 3 with the remaining 20% released by day 14. Bone healing was quantified in rat critical size femoral defects that were treated with hydrogel/PPF and 0, 1, 3, 10, or 30 µg of PTH. Although complete osseous healing was not observed in all samples in any one treatment group, all samples in the 10 µg PTH group were bridged fully by bone or a combination of bone and cartilage containing hypertrophic chondrocytes and endochondral ossification. Outcome measures indicated improved defect bridging by a combination of bony and cartilaginous tissue in the 10 μg treatment group compared with empty bone defects and defects treated with only hydrogel/PPF (i.e., without PTH). Given the tailorability of the hydrogel, future studies will investigate the effects of prolonged gradual PTH release on bone healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:536-544, 2020.
Enhanced Skull Bone Regeneration by Sustained Release of BMP-2 in Interpenetrating Composite Hydrogels.
Kim Sungjun,Kim Junhyung,Gajendiran Mani,Yoon Minhyuk,Hwang Mintai P,Wang Yadong,Kang Byung-Jae,Kim Kyobum
Direct administration of bone morphogenetic protein-2 (BMP-2) for bone regeneration could cause various clinical side effects such as osteoclast activation, inflammation, adipogenesis, and bone cyst formation. In this study, thiolated gelatin/poly(ethylene glycol) diacrylate (PEGDA) interpenetrating (IPN) composite hydrogels were developed for guided skull bone regeneration. To promote bone regeneration, either polycation-based coacervates (Coa) or gelatin microparticles (GMPs) were incorporated within IPN gels as BMP-2 carriers. Both BMP-2 loaded Coa and BMP-2 loaded GMPs showed significantly enhanced in vitro alkaline phosphate (ALP) activity of human mesenchymal stem cells (hMSCs) than non-BMP-2 treated control. Moreover, BMP-2 loaded GMPs group exhibited statistically increased ALP activity compared to both bolus BMP-2 administration and BMP-2 loaded Coa group, indicating that our carriers could protect and maintain biological activity of cargo BMP-2. Sustained release kinetics of BMP-2 from IPN composite hydrogels could be controlled by different formulations. For in vivo bone regeneration, various IPN gel formulations (i.e., (1) control, (2) only hydrogel, (3) hydrogel with bolus BMP-2, (4) hydrogel with BMP-2-loaded Coa, and (5) hydrogel with BMP-2-loaded GMPs) were bilaterally implanted into 5 mm-sized rat calvarial defects. After 4 weeks, micro-CT and histological analysis were performed to evaluate new bone formation. Significantly higher scores for bony bridging and union were observed in BMP-2-loaded Coa and BMP-2-loaded GMP groups as compared to other formulations. In addition, rats treated with BMP-2-loaded GMPs showed a significantly higher ratio of bone volume/total volume and lower trabecular separation scores than others. Finally, rats treated with either Coa or GMP groups exhibited a significant increase in bone formation area, as assessed via histomorphometric analysis. Taken together, it could be concluded that Coa and GMPs were effective carriers to maintain the bioactivity of cargo BMP-2 during its sustained release. Consequently, our IPN composite hydrogel system that combines such BMP-2 carriers could effectively promote skull bone regeneration.
Hypoxia mimicking hydrogels to regulate the fate of transplanted stem cells.
Sathy Binulal N,Daly Andrew,Gonzalez-Fernandez Tomas,Olvera Dinorath,Cunniffe Grainne,McCarthy Helen O,Dunne Nicholas,Jeon Oju,Alsberg Eben,Donahue Tammy L Haut,Kelly Daniel J
Controlling the phenotype of transplanted stem cells is integral to ensuring their therapeutic efficacy. Hypoxia is a known regulator of stem cell fate, the effects of which can be mimicked using hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors such as dimethyloxalylglycine (DMOG). By releasing DMOG from mesenchymal stem cell (MSC) laden alginate hydrogels, it is possible to stabilize HIF-1α and enhance its nuclear localization. This correlated with enhanced chondrogenesis and a reduction in the expression of markers associated with chondrocyte hypertrophy, as well as increased SMAD 2/3 nuclear localization in the encapsulated MSCs. In vivo, DMOG delivery significantly reduced mineralisation of the proteoglycan-rich cartilaginous tissue generated by MSCs within alginate hydrogels loaded with TGF-β3 and BMP-2. Together these findings point to the potential of hypoxia mimicking hydrogels to control the fate of stem cells following their implantation into the body. STATEMENT OF SIGNIFICANCE: There are relatively few examples where in vivo delivery of adult stem cells has demonstrated a true therapeutic benefit. This may be attributed, at least in part, to a failure to control the fate of transplanted stem cells in vivo. In this paper we describe the development of hydrogels that mimic the effects of hypoxia on encapsulated stem cells. In vitro, these hydrogels enhance chondrogenesis of MSCs and suppress markers associated with chondrocyte hypertrophy. In an in vivo environment that otherwise supports progression along an endochondral pathway, we show that these hydrogels will instead direct mesenchymal stem cells (MSCs) to produce a more stable, cartilage-like tissue. In addition, we explore potential molecular mechanisms responsible for these phenotypic changes in MSCs.
The Cocktail Effect of BMP-2 and TGF-β1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model.
Yoon Sun-Jung,Yoo Youngbum,Nam Sang Eun,Hyun Hoon,Lee Deok-Won,Um Sewook,Kim So Yeon,Hong Sung Ok,Yang Dae Hyeok,Chun Heung Jae
Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-β1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-β1 (10 ng) (GC/BMP-2/TGF-β1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-β1 (5 ng) (GC/BMP-2/TGF-β1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-β1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.
Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies.
Nam Sungmin,Stowers Ryan,Lou Junzhe,Xia Yan,Chaudhuri Ovijit
Hydrogels are commonly used as artificial extracellular matrices for 3D cell culture and for tissue engineering. Viscoelastic hydrogels with tunable stress relaxation have recently been developed, and stress relaxation in the hydrogels has been found to play a key role in regulating cell behaviors such as differentiation, spreading, and proliferation. Here we report a simple but precise materials approach to tuning stress relaxation of alginate hydrogels with polyethylene glycol (PEG) covalently grafted onto the alginate. Hydrogel relaxation was modulated independent of the initial elastic modulus by varying molecular weight and concentration of PEG along with calcium crosslinking of the alginate. Increased concentration and molecular weight of the PEG resulted in faster stress relaxation, a higher loss modulus, and increased creep. Interestingly, we found that stress relaxation of the hydrogels is determined by the total mass amount of PEG in the hydrogel, and not the molecular weight or concentration of PEG chains alone. We then evaluated the utility of these hydrogels for 3D cell culture. Faster relaxation in RGD-coupled alginate-PEG hydrogels led to increased spreading and proliferation of fibroblasts, and enhanced osteogenic differentiation of mesenchymal stem cells (MSCs). Thus, this work establishes a new materials approach to tuning stress relaxation in alginate hydrogels for 3D cell culture.
Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle.
Shekaran Asha,García José R,Clark Amy Y,Kavanaugh Taylor E,Lin Angela S,Guldberg Robert E,García Andrés J
Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.
The effects of hydroxyapatite nanoparticles embedded in a MMP-sensitive photoclickable PEG hydrogel on encapsulated MC3T3-E1 pre-osteoblasts.
Carles-Carner Maria,Saleh Leila S,Bryant Stephanie J
Biomedical materials (Bristol, England)
This study investigated the effects of introducing hydroxyapatite nanoparticles into a matrix metalloproteinase (MMP) sensitive poly(ethylene glycol) (PEG) hydrogel containing cell adhesion peptides of RGD for bone tissue engineering. MC3T3-E1 pre-osteoblasts were encapsulated in the biomimetic PEG hydrogel, which was formed from the photoclick thiol-norbornene reaction system, cultured for up to 28 d in growth medium or osteogenic differentiation medium, and evaluated by cellular morphology and differentiation by alkaline phosphatase (ALP) activity and bone-like extracellular matrix deposition for mineral and collagen. Hydroxyapatite nanoparticles were incorporated during hydrogel formation and cell encapsulation at 0%, 0.1% or 1% (w/w). Incorporation of hydroxyapatite nanoparticles did not affect the hydrogel properties as measured by compressive modulus and equilibrium swelling. In growth medium, encapsulated MC3T3-E1 cells remained largely round regardless of hydroxyapatite concentration. ALP activity increased by 25% at day 14 and total collagen content increased by 55% at day 28 with increasing hydroxyapatite concentration from 0% to 1%. In differentiation medium, cell spreading was evident regardless of hydroxyapatite indicating that the MC3T3-E1 cells were able to degrade the hydrogel. For the 1% hydroxyapatite condition, ALP activity was 27% higher at day 14 and total collagen content was 22% higher at day 28 in differentiation medium when compared to growth medium. Mineral deposits were more abundant and spatial elaboration of collagen type I was more evident in the 1% (w/w) hydroxyapatite condition with differentiation medium when compared to all other conditions. Overall, osteogenesis was observed in the hydrogels with hydroxyapatite nanoparticles in growth medium but was enhanced in differentiation medium. In summary, a biomimetic hydrogel comprised of MMP-sensitive crosslinks, RGD cell adhesion peptides, and 1% (w/w) hydroxyapatite nanoparticles is promising for bone tissue engineering.
Drug-Loaded Elastin-Like Polypeptide-Collagen Hydrogels with High Modulus for Bone Tissue Engineering.
Pal Pallabi,Nguyen Quynh C,Benton Angela H,Marquart Mary E,Janorkar Amol V
Emphasizing the role of hydrogel stiffness and cellular differentiation, this study develops collagen and elastin-like polypeptide (ELP)-based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) and doxycycline with mechanical properties suitable for osteogenesis. The drug-incorporated collagen-ELP hydrogels has significantly higher modulus of 35 ± 5 kPa compared to collagen-only hydrogels. Doxycycline shows a bi-phasic release with an initial burst release followed by a gradual release, while rhBMP-2 exhibits a nearly linear release profile for all hydrogels. The released doxycycline shows anti-microbial activity against Pseudomonas aeruginosa, Streptococcus sanguinis, and Escherichia coli. Microscopic observation of the hydrogels reveals their interconnected, macroporous, 3D open architecture with pore diameters between 160 and 400 µm. This architecture supports human adipose-derived stem cell attachment and proliferation from initial days of cell seeding, forming a thick cellular sheath by day 21. Interestingly, in collagen and collagen-ELP hydrogels, cell morphology is elongated with stretched slender lamellipodial formation, while cells assemble as spheroidal aggregates in crosslinked as well as drug-loaded hydrogels. Osteogenic markers, alkaline phosphatase and osteocalcin, are expressed maximally for drug-loaded hydrogels compared to those without drugs. The drug-loaded collagen-ELP hydrogels are thus promising for combating bacterial infection and promoting guided bone regeneration.
Self-assembling peptide hydrogels modulate in vitro chondrogenesis of bovine bone marrow stromal cells.
Kopesky Paul W,Vanderploeg Eric J,Sandy John S,Kurz Bodo,Grodzinsky Alan J
Tissue engineering. Part A
Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.
Effect of sustained release of rhBMP-2 from dried and wet hyaluronic acid hydrogel carriers compared with direct dip coating of rhBMP-2 on peri-implant osteogenesis of dental implants in canine mandibles.
Pan Hui,Han Jeong Joon,Park Yong-Doo,Cho Tae Hyung,Hwang Soon Jung
Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery
Hyaluronic acid (HA) hydrogel has been used as a carrier of recombinant human bone morphogenetic protein (rhBMP)-2 for sustained delivery. To enhance peri-implant osteogenesis, a dried coating of rhBMP-2 HA hydrogel (BMP-HAH) on dental implants was designed; this approach provides the advantage of omitting in situ preparation of wet HA hydrogel. Sustained release of rhBMP-2 was more efficient for dried hydrogel over wet hydrogel. For both types, the released rhBMP-2 consistently led to enhanced alkaline phosphatase activity and osterix expression in human mesenchymal stromal cells. Histomorphometric analysis 4 weeks after placement of a dental implant in canine mandibles showed that the dried coating of BMP-HAH (10 μg/ml, n = 6) resulted in a significantly greater bone area (BA) than the wet BMP-HAH (10 μg/ml, n = 6) (p = 0.006) and implants without any coating (n = 6) (p = 0.022), while simple dip coating with rhBMP-2 (10 μg/ml, n = 6) resulted in significantly greater BA than the other three groups (p < 0.0005). Bone-to-implant contact (BIC) was significantly different only between the dried and wet coating of BMP-HAH (p = 0.014). Our results suggest that a simple dip coating of rhBMP-2 is more effective for increased peri-implant osteogenesis compared to a coating of BMP-HAH with sustained release.
Injectable mineralized microsphere-loaded composite hydrogels for bone repair in a sheep bone defect model.
Ingavle Ganesh C,Gionet-Gonzales Marissa,Vorwald Charlotte E,Bohannon Laurie K,Clark Kaitlin,Galuppo Larry D,Leach J Kent
The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.
A pH-Triggered, Self-Assembled, and Bioprintable Hybrid Hydrogel Scaffold for Mesenchymal Stem Cell Based Bone Tissue Engineering.
Zhao Chen,Qazvini Nader Taheri,Sadati Monirosadat,Zeng Zongyue,Huang Shifeng,De La Lastra Ana Losada,Zhang Linghuan,Feng Yixiao,Liu Wei,Huang Bo,Zhang Bo,Dai Zhengyu,Shen Yi,Wang Xi,Luo Wenping,Liu Bo,Lei Yan,Ye Zhenyu,Zhao Ling,Cao Daigui,Yang Lijuan,Chen Xian,Athiviraham Aravind,Lee Michael J,Wolf Jennifer Moriatis,Reid Russell R,Tirrell Matthew,Huang Wei,de Pablo Juan J,He Tong-Chuan
ACS applied materials & interfaces
Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.
An injectable cationic hydrogel electrostatically interacted with BMP2 to enhance in vivo osteogenic differentiation of human turbinate mesenchymal stem cells.
Kim Mal Geum,Kang Tae Woong,Park Joon Yeong,Park Seung Hun,Ji Yun Bae,Ju Hyeon Jin,Kwon Doo Yeon,Kim Young Sik,Kim Sung Won,Lee Bong,Choi Hak Soo,Lee Hai Bang,Kim Jae Ho,Lee Bun Yeoul,Min Byoung Hyun,Kim Moon Suk
Materials science & engineering. C, Materials for biological applications
We have designed and characterized an injectable, electrostatically bonded, in situ-forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(ε-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ-forming hydrogel systems consisting of MP-NH and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8 weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ-forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.
Injectable polypeptide hydrogel/inorganic nanoparticle composites for bone tissue engineering.
Huang Wei-Shun,Chu I-Ming
The general concept of tissue engineering is to restore biological function by replacing defective tissues with implantable, biocompatible, and easily handleable cell-laden scaffolds. In this study, osteoinductive and osteoconductive super paramagnetic Fe3O4 nanoparticles (MNP) and hydroxyapatite (HAP) nanoparticles were incorporated into a di-block copolymer based thermo-responsive hydrogel, methoxy(polyethylene glycol)-polyalanine (mPA), at various concentrations to afford composite, injectable hydrogels. Incorporating nanoparticles into the thermo-responsive hydrogel increased the complex viscosity and decreased the gelation temperature of the starting hydrogel. Functionally, the integration of inorganic nanoparticles modulated bio-markers of bone differentiation and enhanced bone mineralization. Moreover, this study adopted the emerging method of using either a supplementary static magnetic field (SMF) or a moving magnetic field to elicit biological response. These results demonstrate that combining external (magnet) and internal (scaffold) magnetisms is a promising approach for bone regeneration.
Modulating hydrogel crosslink density and degradation to control bone morphogenetic protein delivery and in vivo bone formation.
Holloway Julianne L,Ma Henry,Rai Reena,Burdick Jason A
Journal of controlled release : official journal of the Controlled Release Society
Bone morphogenetic proteins (BMPs) show promise in therapies for improving bone formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on bone repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1-5wt.%) or maleimide functionalization (10-40%f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of bone repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new bone tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, bone volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation.
Transgenic PDGF-BB/sericin hydrogel supports for cell proliferation and osteogenic differentiation.
Wang Feng,Hou Kai,Chen Wenjing,Wang Yuancheng,Wang Riyuan,Tian Chi,Xu Sheng,Ji Yanting,Yang Qianqian,Zhao Ping,Yu Ling,Lu Zhisong,Zhang Huijie,Li Fushu,Wang Han,He Baicheng,Kaplan David L,Xia Qingyou
Sericin has been exploited as a biomaterial due to its biocompatibility, biodegradability, and low-immunogenicity as an isolated polymer and support for cell adhesion. In the present study, human platelet-derived growth factor (PDGF-BB)-functionalized sericin hydrogels were generated using transgenic silkworms, where the as-spun silk incorporated engineered PDGF-BB (termed PDGFM) in the sericin layers of the cocoons. Sericin and PDGFM were simultaneously extracted from the silk fibroin cocoon fibers, and the soluble extract was then formed into a hydrogel via thermal exposure. The PDGFM sericin hydrogels exhibited increased β-sheet content and a compressive modulus of 74.91 ± 2.9 kPa comparable to chemically crosslinked sericin hydrogels (1.68-55.53 kPa) and a porous microstructure, which contributed to cell adhesion and growth. A 13.1% of total extracted PDGFM from the initial silk fibers was incorporated and immobilized in the sericin hydrogels during material processing, and 1.33% of PDGFM was released over 30 days from the hydrogels in vitro. The remaining PDGFM achieved long-term storage/stability in the sericin hydrogels for more than 42 days at 37 °C. In addition, the PDGFM sericin hydrogels were not immunogenic, were biocompatible and bioactive in promoting the support of cell proliferation. When combined with BMP-9, the PDGFM sericin hydrogels provided synergy to support the osteoblastic differentiation of mesenchymal stem cells (hMSCs) in vitro and in vivo. This study demonstrates that genetically functionalized PDGFM sericin hydrogels can provide useful biomaterials to support cell and tissue outcomes, here with a focus on osteogenesis.
Interleukin-4-loaded hydrogel scaffold regulates macrophages polarization to promote bone mesenchymal stem cells osteogenic differentiation via TGF-β1/Smad pathway for repair of bone defect.
Zhang Jiankang,Shi Haitao,Zhang Nian,Hu Liru,Jing Wei,Pan Jian
OBJECTIVE:Tissue engineering is a promising strategy for repair of large bone defect. However, the immune system reactions to biological scaffold are increasingly being recognized as a crucial factor influencing regeneration efficacy. In this study, a bone-bioactive hydrogel bead loaded with interleukin-4 (IL-4) was used to regulate macrophages polarization and accelerate bone regeneration. METHODS:IL-4-loaded calcium-enriched gellan gum (Ca-GG + IL-4) hydrogel beads were synthesised. And the effect on cell behaviour was detected. Furthermore, the effect of the Ca-GG + IL-4 hydrogel bead on macrophage polarization and the effect of macrophage polarization on bone mesenchymal stem cells (BMSCs) apoptosis and osteogenic differentiation were evaluated in vitro and in vivo. RESULTS:BMSCs were able to survive in the hydrogel regardless of whether IL-4 was incorporated. Immunofluorescence staining and qPCR results revealed that Ca-GG + IL-4 hydrogel bead could promote M2 macrophage polarization and increase transforming growth factor (TGF)-β1 expression level, which activates the TGF-β1/Smad signalling pathway in BMSCs and promotes osteogenic differentiation. Moreover, immunohistochemical analysis demonstrated Ca-GG + IL-4 hydrogel bead could promote M2 macrophage polarization and reduce cell apoptosis in vivo. In addition, micro-CT and immunohistochemical analysis at 12 weeks post-surgery showed that Ca-GG + IL-4 hydrogel bead could achieve superior bone defect repair efficacy in vivo. CONCLUSIONS:The Ca-GG + IL-4 hydrogel bead effectively promoted bone defect regeneration via regulating macrophage polarization, reducing cell apoptosis and promoting BMSCs osteogenesis through TGF-β1/Smad pathway. Therefore, it is a promising strategy for repair of bone defect.
Intraosseous Delivery of Bone Morphogenic Protein-2 Using a Self-Assembling Peptide Hydrogel.
Phipps Matthew C,Monte Felipe,Mehta Manav,Kim Harry K W
Osteonecrosis of the femoral head (ONFH) is a debilitating hip disorder, which often produces a permanent femoral head deformity and osteoarthritis. The local delivery of biological agents capable of stimulating bone healing offer potential new treatment options for patients with ONFH. Previous studies from our laboratory have shown that a local intraosseous infusion of bone morphogenic protein-2 (BMP-2) was effective in stimulating new bone formation in a piglet model of ischemic ONFH. However, infusion of BMP-2 solution was associated with unwanted dissemination of BMP-2 out of the femoral head and produced heterotopic ossification in the hip capsule. Injectable hydrogels offer a potential method to control the dissemination of biological molecules in vivo. In the present study, we evaluated the potential of a peptide-based, self-assembling hydrogel called RADA16 to transition from a solution to a gel following infusion into the femoral head, thereby preventing backflow, as well as its potential use as a delivery vehicle for BMP-2. Cadaver pig femoral heads were used to study the backflow and the distribution of RADA16 following an intraosseous infusion. Microcomputed tomography analysis following the infusion of RADA16 mixed with a radiocontrast agent revealed a significant decrease in the amount of back flow of radiocontrast agent down the needle track compared to the soluble infusion of radiocontrast without RADA16. Furthermore, RADA16 mixed with radiocontrast agent showed good distribution within the femoral head. In addition, in vitro experiments revealed that higher concentrations of RADA16 decreased the rate of BMP-2 dissemination out of the hydrogel. The BMP-2 that was released from RADA16 maintains its biological activity, inducing the phosphorylation of SMAD1/5/8 in pig primary bone marrow stromal cells. Lastly, pig primary bone marrow stromal cells showed significantly increased in vitro proliferation on RADA16 hydrogels over 1 week compared to tissue culture plastic, suggesting that it is a suitable matrix for supporting cellular proliferation. In conclusion, RADA16 showed potential for use as a drug delivery vehicle to control the delivery of BMP-2 within the femoral head. This novel therapy may be able to provide benefits to patients suffering from debilitating conditions such as osteonecrosis of the femoral head.
Enhanced osteogenesis of human mesenchymal stem cells by periodic heat shock in self-assembling peptide hydrogel.
Chen Jing,Shi Zhong-Dong,Ji Xinying,Morales Jorge,Zhang Jingwei,Kaur Navneet,Wang Sihong
Tissue engineering. Part A
The mechanisms for the heat-induced osteogenesis are not completely known and the thermal regulation of human mesenchymal stem cell (hMSC) differentiation is not well studied. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into osteoblasts in self-assembling peptide hydrogel and on tissue culture plates were investigated. hMSCs isolated from human bone marrow were seeded in conventional culture plates (two-dimensional [2D] culture) and on the surface of three-dimensional (3D) PuraMatrix peptide hydrogel (3D culture), followed by 1 h HS at 41°C once a week during osteogenic differentiation. Alkaline phosphatase (ALP) activity was enhanced in both 2D and 3D cultures via periodic HS at early stage of differentiation; meanwhile, HS significantly increased the calcium deposition at day 19 and 27 of differentiation in both 2D and 3D cultures. The periodic HS also upregulated osteo-specific genes, osterix (OSX) on day 11, osteopontin (OP) on day 19, and bone morphogenetic protein 2 (BMP2) on day 25 in 2D culture. In 3D PuraMatrix culture, the runt-related transcription factor 2 (Runx2) was upregulated by HS on day 25 of differentiation. The heat shock protein 70 (HSP70) was significantly upregulated by HS in differentiated hMSCs analyzed at 24 h after HS. These results demonstrate that HS induced an earlier differentiation of hMSCs and enhanced the maturation of osteoblasts differentiated from hMSCs. Therefore, mild HS treatment may be potentially used to enhance the bone regeneration using hMSCs. Our data will guide the design of in vivo heating protocols and enable further investigations in thermal treatments of MSC osteogenesis for bone tissue engineering.
Dual Effect of Curcumin/BMP-2 Loaded in HA/PLL Hydrogels on Osteogenesis In Vitro and In Vivo.
Kim Eun-Cheol,Yoon Sun Jung,Noh Kwantae,Lee Deok-Won
Journal of nanoscience and nanotechnology
In the present study, we evaluated the potential of poly-l-lysine/hyaluronic acid (HA/PLL) hydrogels containing curcumin (CUR) and bone morphogenetic protein-2 (BMP-2) as bone tissue regeneration scaffolds. Hydrogels HP-1˜2 were formed by amide bonds via the condensation reactions between 0.02 μmol HA and 0.06–0.12 μmol poly-l-lysine · hydrobromide (PLL · HBr). Physical, chemical, and thermal analyses revealed that the amount of PLL · HBr significantly influenced hydrogel properties. Based on an In Vitro MG-63 cell proliferation test, HP-1˜2 were cytocompatible, and all hydrogels containing different amounts of CUR and BMP-2, except for HA0.02/PLL0.06/CUR20/BMP-2100 (HPCB-4), resulted in cell proliferation above 80%. An In Vitro release test showed that CUR and BMP-2 were consistently released from HA0.02/PLL0.06/CUR15 (HPC), HA0.02/PLL0.06/BMP-2100 (HPB), HA0.02/PLL0.06/CUR15/BMP-210 , 50 , or 100 (HPCB-1˜3), and HA0.02/PLL0.06/CUR10 or 20/BMP-2100 (HPCB-4˜5) for 7 and 28 days, respectively. In Vitro ALP activity and calcium deposition and In Vivo micro-computed tomography (micro-CT) tests demonstrated the potential application of HPCB-3 as bone tissue regeneration scaffolds, suggesting that bone tissue regeneration can be optimized by controlling the amounts of CUR and BMP-2.
Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects.
Park Hyun-Ji,Jin Yoonhee,Shin Jisoo,Yang Kisuk,Lee Changhyun,Yang Hee Seok,Cho Seung-Woo
Over the last few decades, stem cell therapies have been highlighted for their potential to heal damaged tissue and aid in tissue reconstruction. However, materials used to deliver and support implanted cells often display limited efficacy, which has resulted in delaying translation of stem cell therapies into the clinic. In our previous work, we developed a mussel-inspired, catechol-functionalized hyaluronic acid (HA-CA) hydrogel that enabled effective cell transplantation due to its improved biocompatibility and strong tissue adhesiveness. The present study was performed to further expand the utility of HA-CA hydrogels for use in stem cell therapies to treat more clinically relevant tissue defect models. Specifically, we utilized HA-CA hydrogels to potentiate stem cell-mediated angiogenesis and osteogenesis in two tissue defect models: critical limb ischemia and critical-sized calvarial bone defect. HA-CA hydrogels were found to be less cytotoxic to human adipose-derived stem cells (hADSCs) in vitro compared to conventional photopolymerized HA hydrogels. HA-CA hydrogels also retained the angiogenic functionality of hADSCs and supported osteogenic differentiation of hADSCs. Because of their superior tissue adhesiveness, HA-CA hydrogels were able to mediate efficient engraftment of hADSCs into the defect regions. When compared to photopolymerized HA hydrogels, HA-CA hydrogels significantly enhanced hADSC-mediated therapeutic angiogenesis (promoted capillary/arteriole formation, improved vascular perfusion, attenuated ischemic muscle degeneration/fibrosis, and reduced limb amputation) and bone reconstruction (mineralized bone formation, enhanced osteogenic marker expression, and collagen deposition). This study proves the feasibility of using bioinspired HA-CA hydrogels as functional biomaterials for improved tissue regeneration in critical tissue defects.
Efficacy of a Self-Assembling Peptide Hydrogel, SPG-178-Gel, for Bone Regeneration and Three-Dimensional Osteogenic Induction of Dental Pulp Stem Cells.
Tsukamoto Jun,Naruse Keiko,Nagai Yusuke,Kan Shuhei,Nakamura Nobuhisa,Hata Masaki,Omi Maiko,Hayashi Tatsuhide,Kawai Tatsushi,Matsubara Tatsuaki
Tissue engineering. Part A
The aim of this study was to assess the efficacy of a self-assembling peptide hydrogel as a scaffold for bone regeneration. We used a neutral and injectable self-assembling peptide hydrogel, SPG-178-Gel. Bone defects (5 mm in diameter) in rat calvarial bones were filled with a mixture of alpha-modified Eagle's medium and peptide hydrogel. Three weeks after surgery, soft X-ray and microcomputed tomography (micro-CT) images of the gel-treated bones showed new bone formations in the periphery and in central areas of the defects. Next, we evaluated the three-dimensional osteogenic induction of dental pulp stem cells (DPSCs), a type of mesenchymal stem cell, in SPG-178-Gel. We first confirmed that the osteogenic differentiation of DPSCs was significantly promoted by osteogenic induction medium containing recombinant human bone morphogenetic protein-4 (rhBMP-4) in a two-dimensional cell culture. Then, we verified DPSC proliferation and osteogenic differentiation in a three-dimensional cell culture using SPG-178-Gel. The gene expression levels of osteopontin, osteocalcin, and collagen type I were significantly increased when DPSCs were cultured in SPG-178-Gel with the osteogenic induction medium. Micro-CT observations showed the formation of widespread calcium deposition. In conclusion, SPG-178-Gel was adequately effective as a scaffold and can be a suitable tool for bone formation in vivo and in vitro. These findings suggest that the self-assembling peptide hydrogel, SPG-178-Gel, could be a promising tool for bone tissue engineering.
Presentation of BMP-2 mimicking peptides in 3D hydrogels directs cell fate commitment in osteoblasts and mesenchymal stem cells.
Madl Christopher M,Mehta Manav,Duda Georg N,Heilshorn Sarah C,Mooney David J
Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.
[Early effect of graphene oxide-carboxymethyl chitosan hydrogel loaded with interleukin 4 and bone morphogenetic protein 2 on bone immunity and repair].
Zou Min,Sun Jiachen,Xiang Zhou
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery
Objective:To investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods:GO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days. Results:Generally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. , the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect ( <0.05). Conclusion:The GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation , which provide a new strategy for bone defect repair and immune regulation.
Nano-Fibrous Biopolymer Hydrogels via Biological Conjugation for Osteogenesis.
Chen Huinan,Xing Xiaodong,Jia Yang,Mao Jiahui,Zhang Ziwei,Tan Huaping
Journal of nanoscience and nanotechnology
Nanostructured biopolymer hydrogels have great potential in the field of drug delivery and regenerative medicine. In this work, a nano-fibrous (NF) biopolymer hydrogel was developed for cell growth factors (GFs) delivery and in vitro osteogenesis. The nano-fibrous hydrogel was produced via biological conjugation of streptavidin functionalized hyaluronic acid (HA-Streptavidin) and biotin terminated star-shaped poly(ethylene glycol) (PEG-Biotin). In the present work, in vitro gelation, mechanical properties, degradation and equilibrium swelling of the NF hydrogel were examined. The potential application of this NF gel scaffold in bone tissue engineering was confirmed by encapsulation behavior of osteoblasts. Osteoblasts seeded directly in NF gel scaffold containing cell growth factor, e.g. bone morphogenetic protein 2 (BMP-2), was to mimic the in vivo microenvironment in which cells interface biomaterials and interact with BMP-2. In combination with BMP-2, the NF hydrogel exhibited beneficial effects on osteoblast activity and differentiation, which suggested a promising future for local treatment of pathologies involving bone loss.
Amino acid composition of nanofibrillar self-assembling peptide hydrogels affects responses of periodontal tissue cells in vitro.
Koch Franziska,Wolff Anne,Mathes Stephanie,Pieles Uwe,Saxer Sina S,Kreikemeyer Bernd,Peters Kirsten
International journal of nanomedicine
Background:The regeneration of tissue defects at the interface between soft and hard tissue, eg, in the periodontium, poses a challenge due to the divergent tissue requirements. A class of biomaterials that may support the regeneration at the soft-to-hard tissue interface are self-assembling peptides (SAPs), as their physicochemical and mechanical properties can be rationally designed to meet tissue requirements. Materials and methods:In this work, we investigated the effect of two single-component and two complementary β-sheet forming SAP systems on their hydrogel properties such as nanofibrillar architecture, surface charge, and protein adsorption as well as their influence on cell adhesion, morphology, growth, and differentiation. Results:We showed that these four 11-amino acid SAP (P11-SAP) hydrogels possessed physico-chemical characteristics dependent on their amino acid composition that allowed variabilities in nanofibrillar network architecture, surface charge, and protein adsorption (eg, the single-component systems demonstrated an ~30% higher porosity and an almost 2-fold higher protein adsorption compared with the complementary systems). Cytocompatibility studies revealed similar results for cells cultured on the four P11-SAP hydrogels compared with cells on standard cell culture surfaces. The single-component P11-SAP systems showed a 1.7-fold increase in cell adhesion and cellular growth compared with the complementary P11-SAP systems. Moreover, significantly enhanced osteogenic differentiation of human calvarial osteoblasts was detected for the single-component P11-SAP system hydrogels compared with standard cell cultures. Conclusion:Thus, single-component system P11-SAP hydrogels can be assessed as suitable scaffolds for periodontal regeneration therapy, as they provide adjustable, extracellular matrix-mimetic nanofibrillar architecture and favorable cellular interaction with periodontal cells.
Hydroxyapatite Nanoparticle-Crosslinked Peptide Hydrogels for Three-Dimensional Culture and Differentiation of MC3T3-E1 Osteoblasts.
Xu Yingjie,Wu Xin,Wang Shuyi,Yang Changzhou,Li Ying,Cao Yi
Journal of biomedical nanotechnology
Hydrogels have been widely used to mimic the biochemical and mechanical environments of native extracellular matrices for cell culture and tissue engineering. Among them, self-assembling peptide hydrogels are of special interest thanks to their great biocompatibility, designability and convenient preparation procedures. In pioneering studies, self-assembling peptide hydrogels have been used for the culture of bone marrow cells. However, the low mechanical stability of peptide hydrogels seems to be a drawback for these applications, as bone marrow cells prefer hard substrates for osteogenic differentiation. In this work, we explored the use of hydroxyapatite (HAP)-peptide hybrid hydrogels for three-dimensional (3D) culture and differentiation of osteogenic MC3T3-E1 cells. We used HAP nanoparticles as crosslinkers to increase the mechanical stability of peptide hydrogels. Meanwhile, HAP provided unique chemical cues to promote the differentiation of osteoblasts. A phosphate group was introduced to the self-assembling peptide so that the peptide fibers could bind to HAP nanoparticles specifically and strongly. Rheological characterization indicated that the hybrid hydrogels were mechanically more stable than the hydrogels containing only peptides and can be used for long term cell culture. Moreover, the hydrogels were biocompatible and showed very low cytotoxicity. The favorable mechanical properties of the hybrid hydrogels and the chemical properties of HAP synergistically supported the differentiation of MC3T3-E1 cells. Based on these characterizations, we believe that these hybrid hydrogels can potentially be used as scaffolds for cartilage and bone regeneration in the future.
Hydrogels functionalized with N-cadherin mimetic peptide enhance osteogenesis of hMSCs by emulating the osteogenic niche.
Zhu Meiling,Lin Sien,Sun Yuxin,Feng Qian,Li Gang,Bian Liming
N-cadherin is considered to be the key factor in directing cell-cell interactions during mesenchymal condensation, which is essential to osteogenesis. In this study, hyaluronic acid (HA) hydrogels are biofunctionalized with an N-cadherin mimetic peptide to mimic the pro-osteogenic niche in the endosteal space to promote the osteogenesis of human mesenchymal stem cells (hMSCs). Results show that the conjugation of the N-cadherin peptide in the HA hydrogels enhances the expression of the osteogenic marker genes in the seeded hMSCs. Furthermore, the biofunctionalized HA hydrogels promote the alkaline phosphatase activity, type I collagen deposition, and matrix mineralization by the seeded hMSCs under both in vitro and in vivo condition. We postulate that the biofunctionalized hydrogels emulates the N-cadherin-mediated homotypic cell-cell adhesion among MSCs and the "orthotypic" interaction between the osteoblasts and MSCs. These findings demonstrate that the biofunctionalized HA hydrogels provide a supportive niche microenvironment for the osteogenesis of hMSCs.