Incorporation of a silicon-based polymer to PEG-DA templated hydrogel scaffolds for bioactivity and osteoinductivity.
ABSTRACT: A scaffold that is inherently bioactive, osteoinductive and osteoconductive may guide mesenchymal stem cells (MSCs) to regenerate bone tissue in the absence of exogenous growth factors. Previously, we established that hydrogel scaffolds formed by crosslinking methacrylated star poly(dimethylsiloxane) (PDMSstar-MA) with diacrylated poly(ethylene glycol) (PEG-DA) promote bone bonding by induction of hydroxyapatite formation ("bioactive") and promote MSC lineage progression toward osteoblast-like fate ("osteoinductive"). Herein, we have combined solvent induced phase separation (SIPS) with a fused salt template to create PDMSstar-PEG hydrogel scaffolds with controlled PDMSstar-MA distribution as well as interconnected macropores of a tunable size to allow for subsequent cell seeding and neotissue infiltration ("osteoconductive"). Scaffolds were prepared with PDMSstar-MA of two number average molecular weights (Mns) (2k and 7k) with varying PDMSstar-MA:PEG-DA ratios and template salt sizes. The distribution of PDMSstar-MA within the hydrogels was examined as well as pore size, percent interconnectivity, dynamic and static moduli, hydration, degradation and in vitro bioactivity (i.e. mineralization when exposed to simulated body fluid, SBF). Finally, cell culture with seeded human bone marrow-derived MSCs (hBMSCs) was used to confirm non-cytotoxicity and characterize osteoinductivity. Tunable, interconnected macropores were achieved by utilization of a fused salt template of a specified salt size during fabrication. Distribution of PDMSstar-MA within the PEG-DA matrix improved for the lower Mn and contributed to differences in specific material properties (e.g. local modulus) and cellular response. However, all templated SIPS PDMSstar-PEG hydrogels were confirmed to be bioactive, non-cytotoxic and displayed PDMSstar-MA dose-dependent osteogenesis. STATEMENT OF SIGNIFICANCE: A tissue engineering scaffold that can inherently guide mesenchymal stem cells (MSCs) to regenerate bone tissue without growth factors would be a more cost-effective and safe strategy for bone repair. Typically, glass/ceramic fillers are utilized to achieve this through their ability to induce hydroxyapatite formation ("bioactive") and promote MSC differentiation to an osteoblast-like fate ("osteoinductive"). Herein, we have fabricated an interconnected, macroporous PEG-DA hydrogel scaffold that utilizes PDMSstar-MA as a bioactive and osteoinductive scaffold component. We were able to show that these PDMSstar-PEG hydrogels maintain several key material characteristics for bone repair. Further, bioactivity and osteoinductivity were simultaneously achieved in human bone marrow-derived MSC culture, representing a notable achievement for an exclusively material-based strategy.
Project description:In tissue engineering, the physical and chemical properties of the scaffold mediates cell behavior, including regeneration. Thus a strategy that permits rapid screening of cell-scaffold interactions is critical. Herein, we have prepared eight "hybrid" hydrogel scaffolds in the form of continuous gradients such that a single scaffold contains spatially varied properties. These scaffolds are based on combining an inorganic macromer (methacrylated star polydimethylsiloxane, PDMSstar-MA) and organic macromer (poly(ethylene glycol)diacrylate, PEG-DA) as well as both aqueous and organic fabrication solvents. Having previously demonstrated its bioactivity and osteoinductivity, PDMSstar-MA is a particularly powerful component to incorporate into instructive gradient scaffolds based on PEG-DA. The following parameters were varied to produce the different gradients or gradual transitions in: (1) the wt.% ratio of PDMSstar-MA to PEG-DA macromers, (2) the total wt.% macromer concentration, (3) the number average molecular weight (Mn) of PEG-DA and (4) the Mn of PDMSstar-MA. Upon dividing each scaffold into four "zones" perpendicular to the gradient, we were able to demonstrate the spatial variation in morphology, bioactivity, swelling and modulus. Among these gradient scaffolds are those in which swelling and modulus are conveniently decoupled. In addition to rapid screening of cell-material interactions, these scaffolds are well suited for regeneration of interfacial tissues (e.g. osteochondral tissues) that transition from one tissue type to another.
Project description:Inorganic-organic hydrogels with tunable chemical and physical properties were prepared from methacrylated star polydimethylsiloxane (PDMS(star)-MA) and diacrylated poly(ethylene glycol) (PEG-DA) for use as tissue engineering scaffolds. A total of 18 compositionally unique hydrogels were prepared by photo-cross-linking, varying weight ratios of PEG-DA and PDMS(star)-MA of different molecular weights (M(n)): PEG-DA (M(n) = 3.4k and 6k g/mol) and PDMS(star)-MA (M(n) = 1.8k, 5k, and 7k g/mol). Introduction of PDMS(star)-MA caused formation of discrete PDMS-enriched microparticles dispersed within the PEG matrix. The swelling ratio, mechanical properties in tension and compression, nonspecific protein adhesion, controlled introduction of bioactivity, and cytotoxicity of hydrogels were studied. This library of inorganic-organic hydrogels with tunable properties provides a useful platform to study the effect of scaffold properties on cell behavior.
Project description:Inorganic-organic hydrogels based on methacrylated star polydimethylsiloxane (PDMS(star)-MA) and diacrylated poly(ethylene glycol) (PEG-DA) macromers were prepared via solvent-induced phase separation (SIPS). The macromers were combined in a dichloromethane precursor solution and sequentially photopolymerized, dried and hydrated. The chemical and physical properties of the hydrogels were further tailored by varying the number average molecular weight (M(n)) of PEG-DA (M(n)=3.4k and 6k gmol(-1)) as well as the weight percent ratio of PDMS(star)-MA (M(n)=7k gmol(-1)) to PEG-DA from 0:100 to 20:80. Compared to analogous hydrogels fabricated from aqueous precursor solutions, SIPS produced hydrogels with a macroporous morphology, a more even distribution of PDMS(star)-MA, increased modulus and enhanced degradation rates. The morphology, swelling ratio, mechanical properties, bioactivity, non-specific protein adhesion, controlled introduction of cell adhesion, and cytocompatibility of the hydrogels were characterized. As a result of their tunable properties, this library of hydrogels is useful to study material-guided cell behavior and ultimate tissue regeneration.
Project description:Large bone defects and nonunions are serious complications that are caused by extensive trauma or tumour. As traditional therapies fail to repair these critical-sized defects, tissue engineering scaffolds can be used to regenerate the damaged tissue. Highly porous titanium scaffolds, produced by selective laser sintering with mechanical properties in range of trabecular bone (compressive strength 35 MPa and modulus 73 MPa), can be used in these orthopaedic applications, if a stable mechanical fixation is provided. Hydroxyapatite coatings are generally considered essential and/or beneficial for bone formation; however, debonding of the coatings is one of the main concerns. We hypothesised that the titanium scaffolds have an intrinsic potential to induce bone formation without the need for a hydroxyapatite coating. In this paper, titanium scaffolds coated with hydroxyapatite using electrochemical method were fabricated and osteoinductivity of coated and noncoated scaffolds was compared in vitro. Alizarin Red quantification confirmed osteogenesis independent of coating. Bone formation and ingrowth into the titanium scaffolds were evaluated in sheep stifle joints. The examinations after 3 months revealed 70% bone ingrowth into the scaffold confirming its osteoinductive capacity. It is shown that the developed titanium scaffold has an intrinsic capacity for bone formation and is a suitable scaffold for bone tissue engineering.
Project description:The rapid and effective bone regeneration of large non-healing defects remains challenging. Bioactive proteins, such as bone morphogenetic protein (BMP)-2, are proved their osteoinductivity, but their clinical use is currently limited to collagen as biomaterial. Being able to deliver BMP-2 from any other biomaterial would broaden its clinical use. This work presents a novel means for repairing a critical size volumetric bone femoral defect in the rat by combining a osteoinductive surface coating (2D) to a polymeric scaffold (3D hollow tube) made of commercially-available PLGA. Using a polyelectrolyte film as BMP-2 carrier, we tune the amount of BMP-2 loaded in and released from the polyelectrolyte film coating over a large extent by controlling the film crosslinking level and initial concentration of BMP-2 in solution. Using microcomputed tomography and quantitative analysis of the regenerated bone growth kinetics, we show that the amount of newly formed bone and kinetics can be modulated: an effective and fast repair was obtained in 1-2 weeks in the best conditions, including complete defect bridging, formation of vascularized and mineralized bone tissue. Histological staining and high-resolution computed tomography revealed the presence of bone regeneration inside and around the tube with spatially distinct organization for trabecular-like and cortical bones. The amount of cortical bone and its thickness increased with the BMP-2 dose. In view of the recent developments in additive manufacturing techniques, this surface-coating technology may be applied in combination with various types of polymeric or metallic scaffolds to offer new perspectives of bone regeneration in personalized medicine.
Project description:Poly(ethylene glycol) diacrylate (PEG-DA) hydrogels are widely utilized to probe cell-material interactions and ultimately for a material-guided approach to tissue regeneration. In this study, PEG-DA hydrogels were fabricated via solvent-induced phase separation (SIPS) to obtain hydrogels with a broader range of tunable physical properties including morphology (e.g. porosity), swelling and modulus (G'). In contrast to conventional PEG-DA hydrogels prepared from an aqueous precursor solution, the reported SIPS protocol utilized a dichloromethane (DCM) precursor solution which was sequentially photopolymerized, dried and hydrated. Physical properties were further tailored by varying the PEG-DA wt% concentration (5 wt%-25 wt%) and M(n) (3.4k and 6k g mol (-1)). SIPS produced PEG-DA hydrogels with a macroporous morphology as well as increased G' values versus the corresponding conventional PEG-DA hydrogels. Notably, since the total swelling was not significantly changed versus the corresponding conventional PEG-DA hydrogels, pairs or series of hydrogels represent scaffolds in which morphology and hydration or G' and hydration are uncoupled. In addition, PEG-DA hydrogels prepared via SIPS exhibited enhanced degradation rates.
Project description:<h4>Background</h4>Demineralized bone matrix is commonly used as a bone graft substitute, either alone or to supplement an osteoconductive material, because of its osteoinductive properties. The aging of the population has led to an increase in the number of prospective donors of demineralized bone matrix who have taken bisphosphonates to prevent osteoclast-mediated bone resorption. The aim of this study was to determine whether oral bisphosphonate usage affects the osteoinductivity of demineralized bone matrix from donors.<h4>Methods</h4>Sex-matched and age-matched pairs of samples were provided by four tissue banks (three or four pairs per bank). Demineralized bone matrix donors without bisphosphonate treatment had a mean age (and standard deviation) of 69.1 ± 2.5 years, and donors with bisphosphonate treatment had a mean age of 68.9 ± 2.0 years. Each pair included one donor known to have taken bisphosphonates and one who had not taken bisphosphonates. Demineralized bone matrix previously confirmed as osteoinductive was the positive control, and heat-inactivated demineralized bone matrix was the negative control. Demineralized bone matrix incubated with 1 mL of phosphate-buffered saline solution containing 0, 0.002, 2.0, or 2000 ng/mL of alendronate was also tested. Gelatin capsules containing 15 mg of demineralized bone matrix were implanted bilaterally in the gastrocnemius muscle of male nude mice (eight implants per group). The mice were killed thirty-five days after implantation, and hind limbs were recovered and processed for histological analysis. Osteoinductivity was measured with use of a qualitative score and by histomorphometry.<h4>Results</h4>Nine of fifteen samples from donors who had had bisphosphonate treatment and ten of fifteen samples from patients who had not had bisphosphonate treatment were osteoinductive. Qualitative mean scores were comparable (1.7 ± 0.4 for those without bisphosphonates and 1.9 ± 0.7 for those with bisphosphonates). Osteoinductive demineralized bone matrix samples produced ossicles of comparable size, regardless of bisphosphonate usage. Histomorphometric measurements of the area of new bone formation and residual demineralized bone matrix were also comparable. The addition of alendronate to control demineralized bone matrix did not affect its osteoinductivity.<h4>Conclusions</h4>Demineralized bone matrix samples from donors treated with bisphosphonates and donors not treated with bisphosphonates have the same ability to induce bone formation. However, it is not known if the quality of the new bone is affected, with subsequent consequences affecting bone remodeling.
Project description:An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73-92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005-0.005?mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the total concentration.
Project description:The highly tunable biological, chemical, and physical properties of bioactive hydrogels enable their use in an array of tissue engineering and drug delivery applications. Systematic modulation of these properties can be used to elucidate key cell-material interactions to improve therapeutic effects. For example, the rate and extent of endothelialization are critical to the long-term success of many blood-contacting devices. To this end, we have developed a bioactive hydrogel that could be used as coating on cardiovascular devices to enhance endothelial cell (EC) adhesion and migration. The current work investigates the relative impact of hydrogel variables on key endothelialization processes. The bioactive hydrogel is based on poly(ethylene glycol) (PEG) and a streptococcal collagen-like (Scl2-2) protein that has been modified with integrin ?1?1 and ?2?1 binding sites. The use of PEG hydrogels allows for incorporation of specific bioactive cues and independent manipulation of scaffold properties. The selective integrin binding of Scl2-2 was compared to more traditional collagen-modified PEG hydrogels to determine the effect of integrin binding on cell behavior. Protein functionalization density, protein concentration, and substrate modulus were independently tuned with both Scl2-2 and collagen to determine the effect of each variable on EC adhesion, spreading, and migration. The findings here demonstrate that increasing substrate modulus, decreasing functionalization density, and increasing protein concentration can be utilized to increase EC adhesion and migration. Additionally, PEG-Scl2-2 hydrogels had higher migration speeds and proliferation over 1 week compared with PEG-collagen gels, demonstrating that selective integrin binding can be used to enhance cell-material interactions. Overall, these studies contribute to the understanding of the effects of matrix cues on EC interactions and demonstrate the strong potential of PEG-Scl2-2 hydrogels to promote endothelialization of blood-contacting devices.