Mineralized Collagen Scaffolds Research Articles

Mg-Sr-Ca containing bioactive glass nanoparticles hydrogel modified mineralized collagen scaffold for bone repair.

The aim of this study is to explore the therapeutic effects of Mg-Sr-Ca containing bioactive glass nanoparticles sodium alginate hydrogel modified mineralized collagen scaffold (Mg-Sr-Ca-BGNs-SA-MC) on the repair of osteoporotic bone defect. During the study, Mg-Sr-Ca containing bioactive glass nanoparticles (Mg-Sr-Ca-BGNs) were synthesized using the sol-gel method, and the Mg-Sr-Ca-BGNs-SA-MC scaffold was synthesized by a simple method. The Mg-Sr-Ca-BGNs and the Mg-Sr-Ca-BGNs-SA-MC scaffold were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The elements of Mg, Sr, Ca and Si were effectively integrated into Mg-Sr-Ca-BGNs. SEM analysis revealed the presence of Mg-Sr-Ca-BGNs on the scaffold's surface. Furthermore, the cytotoxicity of the scaffolds were assessed using a live/dead assay. The result of the live/dead assay demonstrated that the scaffold materials were non-toxic to cell growth. More importantly, the in vivo study indicated that implanted scaffold promoted tissue regeneration and integration with newly formed bone. Overall, the Mg-Sr-Ca-BGNs-SA-MC scaffold is suitable for guided bone regeneration and beneficial to repair of osteoporotic bone defects.

Evaluating osteogenic effects associated with the incorporation of ascorbic acid in mineralized collagen scaffolds.

Current treatments for craniomaxillofacial (CMF) defects motivate the design of instructive biomaterials that can promote osteogenic healing of complex bone defects. We report methods to promote in vitro osteogenesis of human mesenchymal stem cells (hMSCs) within a model mineralized collagen scaffold via the incorporation of ascorbic acid (vitamin C), a key factor in collagen biosynthesis and bone mineralization. An addition of 5 w/v% ascorbic acid into the base mineralized collagen scaffold significantly changes key morphology characteristics including porosity, macrostructure, and microstructure. This modification promotes hMSC metabolic activity, ALP activity, and hMSC-mediated deposition of calcium and phosphorous. Additionally, the incorporation of ascorbic acid influences osteogenic gene expression (BMP-2, RUNX2, COL1A2) and delays the expression of genes associated with osteoclast activity and bone resorption (OPN, CTSK), though it reduces the secretion of OPG. Together, these findings highlight ascorbic acid as a relevant component for mineralized collagen scaffold design to promote osteogenic differentiation and new bone formation for improved CMF outcomes.

Generative design approach to combine architected Voronoi foams with porous collagen scaffolds to create a tunable composite biomaterial

Regenerative biomaterials for musculoskeletal defects must address multi-scale mechanical challenges. Repairing craniomaxillofacial bone defects, which are often large and irregularly shaped, requires close conformal contact between implant and defect margins to aid healing. While mineralized collagen scaffolds can promote mesenchymal stem cell osteogenic differentiation in vitro and bone formation in vivo, their mechanical performance is insufficient for surgical translation. We report a generative design approach to create scaffold-mesh composites by embedding a macro-scale polymeric Voronoi mesh into the mineralized collagen scaffold. The mechanics of architected foam reinforced composites are defined by a rigorous predictive moduli equation. We show biphasic composites localize strain during loading. Further, planar and 3D mesh-scaffold composites can be rapidly shaped to aid conformal fitting. Voronoi-based composites overcome traditional porosity-mechanics relationship limits while enabling rapid shaping of regenerative implants to conformally fit complex defects unique for individual patients. Statement of SignificanceBiomaterial strategies for (craniomaxillofacial) bone regeneration are often limited by the size and complex geometry of the defects. Voronoi structures are open-cell foams with tunable mechanical properties which have primarily been used computationally. We describe generative design strategies to create Voronoi foams via 3D-printing then embed them into an osteogenic mineralized collagen scaffold to form a multi-scale composite biomaterial. Voronoi structures have predictable and tailorable moduli, permit stain localization to defined regions of the composite, and permit conformal fitting to effect margins to aid surgical practicality and improve host-biomaterial interactions. Multi-scale composites based on Voronoi foams represent an adaptable design approach to address significant challenges to large-scale bone repair.

Inflammatory Licensed hMSCs Exhibit Enhanced Immunomodulatory Capacity in a Biomaterial Mediated Manner.

Craniomaxillofacial (CMF) bone injuries represent particularly challenging environments for regenerative healing due to their large sizes, irregular and unique defect shapes, angiogenic requirements, and mechanical stabilization needs. These defects also exhibit a heightened inflammatory environment that can complicate the healing process. This study investigates the influence of the initial inflammatory stance of human mesenchymal stem cells (hMSCs) on key osteogenic, angiogenic, and immunomodulatory criteria when cultured in a class of mineralized collagen scaffolds under development for CMF bone repair. We previously showed that changes in scaffold pore anisotropy and glycosaminoglycan content can significantly alter the regenerative activity of both MSCs and macrophages. While MSCs are known to adopt an immunomodulatory phenotype in response to inflammatory stimuli, here, we define the nature and persistence of MSC osteogenic, angiogenic, and immunomodulatory phenotypes in a 3D mineralized collagen environment, and further, whether changes to scaffold architecture and organic composition can blunt or accentuate this response as a function of inflammatory licensing. Notably, we found that a one-time licensing treatment of MSCs induced higher immunomodulatory potential compared to basal MSCs as observed by sustained immunomodulatory gene expression throughout the first 7 days as well as an increase in immunomodulatory cytokine (PGE2 and IL-6) expression throughout a 21-day culture period. Further, heparin scaffolds facilitated higher osteogenic cytokine secretion but lower immunomodulatory cytokine secretion compared to chondroitin-6-sulfate scaffolds. Anisotropic scaffolds facilitated higher secretion of both osteogenic protein OPG and immunomodulatory cytokines (PGE2 and IL-6) compared to isotropic scaffolds. These results highlight the importance of scaffold properties on the sustained kinetics of cell response to an inflammatory stimulus. The development of a biomaterial scaffold capable of interfacing with hMSCs to facilitate both immunomodulatory and osteogenic responses is an essential next step to determining the quality and kinetics of craniofacial bone repair.

Synergistic intrafibrillar/extrafibrillar mineralization of collagen fibrils and scaffolds enhanced by introducing polyacrylamide to PILP for osteogenic differentiation

AbstractMineralization can help improve the mechanical properties and degradation rates of collagen scaffolds while ensuring good biocompatibility, thereby providing a suitable microenvironment for osteogenic differentiation. Intrafibrillar/extrafibrillar mineralization of collagen is promoted by polymer‐induced liquid precursors (PILP). In this study, polyacrylamide (PAM) was introduced to PILP which synergistically mineralized collagen scaffold intrafibrillarly and extrafibrillarly. PAM and polyacrylic acid (PAA) can stabilize the amorphous calcium phosphate (ACP), to form a PILP of PAM and PAA/ACP. As a control, another scaffold material was formed using the conventional mineralization method, that is, soaking collagen in a simulated body fluid. Collagen mineralization was characterized using SEM and TEM. After 7 days of mineralization with PILP, intrafibrillar crystallization of collagen was significantly higher than that in the control group, and the stiffness and modulus of this scaffold material significantly increased. Cellular experiment results indicated that the PILP‐mineralized collagen scaffold was biocompatible and promoted the osteogenic differentiation of MC3T3‐E1 pre‐osteoblasts. Biomimetic mineralization by PILP will assist the fabrication of mineralized collagen scaffold for bone repair applications.

Abstract 3660: Bone mechanics influence cancer stem cell formation in osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary tumor of bone and is characterized by rapid bone remodeling accompanied by structural and mechanical heterogeneity across the tumor. Chemoresistance and recurrence of OS can be attributed to a subpopulation of OS cells known as cancer stem-like cells (OS-CSCs). It can be hypothesized that tumor niches possessing distinct structural and mechanical properties could be harboring different OS cell populations, and certain niches might be more conducive for OS-CSCs formation and maintenance. To evaluate the presence of OS-CSCs, mineralized collagen scaffolds mimicking heterogenous bone niches with distinct structural and mechanical properties were fabricated by changing collagen content and pore alignment and were cultured with the highly metastatic human OS cell line 143B. Scaffolds with 47% collagen by weight but different pore alignment were chosen for further analysis because they possessed different stiffness and topographical properties. Cytoskeletal changes, epigenetic changes, chemoresistance and chemoresistance-related pathways, and the levels of stemness genes were assessed in the 3D scaffold cultures. A consistent trend of OS-CSC phenotype was observed in the cell population grown in the softer bone niches (12 kPa) with aligned pores as compared to stiffer niche (31 kPa) with non-aligned pores. The cell population growing in softer, aligned scaffolds had a significantly higher expression of stemness genes (OCT4, NANOG, SOX2, ALDH1A1) and drug efflux gene ABCB1 and increased chemoresistance to doxorubicin and cisplatin. Epigenetic changes associated with cisplatin resistance in OS such as a decrease in methyl transferase EZH2 accompanied by the upregulation of LIF/Notch and PRKCA pathways was observed in cells grown in softer, aligned niches. Fluorescent imaging of actin organization showed rounder cells in the aligned niche indicative of the CSC phenotype, and stretched, elongated cells in the non-aligned niche indicative of a differentiated phenotype. Our study showed that the mechanical and structural properties of bone niches influence the formation of distinct OS cell populations. A softer niche with an aligned structure is more conducive for maintaining OS-CSCs. Citation Format: Zunaira Shoaib, Aleczandria S. Tiffany, Brendan Harley, Joseph Irudayaraj, Timothy M. Fan. Bone mechanics influence cancer stem cell formation in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3660.

Biomimetic mineralized collagen scaffolds enhancing odontogenic differentiation of hDPSCs and dentin regeneration through modulating mechanical microenvironment

Dentin regeneration is a huge challenge because of its complicated structure and the shortage of odontoblasts. Unlike in vitro remineralization of dentin through supersaturation of calcium phosphate, the materials of mineralized collagen scaffolds could recruit the human dental pulp stem cells (hDPSCs) and induce their differentiation in vivo and achieve dentin repair. Inspired by the biomineralization of dentin, herein, mineralized collagen fibrils with various mineralization degrees and mechanical properties were prepared through biomolecule hyaluronic acid (HA) modification, and first time find the mechanical properties have a positive correlation with mineralization degree. When 1.2 mg/mL HA was used to modification, the mineralized HA-modified collagen scaffolds (HA-MCS) with similar modulus and stiffness to that of natural dentin could be obtained. Furthermore, testing ALP activity and alizarin red S staining experiments confirmed the HA-MCS with a biomimetic mechanical microenvironment could enhance the proliferation and differentiation of hDPSCs. Additionally, in vivo experiments performed on the beagle model further proved this point that HA-MCS with a biomimetic mechanical microenvironment could promote dentin regeneration. These results not only elucidate that the modulation of mechanical properties of biomaterials could enhance the proliferation and differentiation of stem cells, but also provide a strategy for hard tissue regeneration.

Combined Application of BMP-2 and Naturally Occurring Bioactive Factor Mixtures for the Optimized Therapy of Segmental Bone Defects.

Critical bone defects are the result of traumatic, infection- or tumor-induced segmental bone loss and represent a therapeutic problem that has not been solved by current reconstructive or regenerative strategies yet. Scaffolds functionalized with naturally occurring bioactive factor mixtures show a promising chemotactic and angiogenic potential in vitro and therefore might stimulate bone regeneration in vivo. To assess this prospect, the study targets at heparin-modified mineralized collagen scaffolds functionalized with naturally occurring bioactive factor mixtures and/or rhBMP-2. These scaffolds were implanted into a 2-mm segmental femoral defect in mice and analyzed in respect to newly formed bone volume (BV) and bone mineral density (BMD) by micro-computed tomography scans after an observation period of 6 weeks. To rate the degree of defect healing, the number of vessels, and the activity of osteoclasts and osteoblasts were analyzed histologically. The sole application of bioactive factor mixtures is inferior to the use of the recombinant growth factor rhBMP-2 regarding BV and degree of defect healing. A higher rhBMP-2 concentration or the combination with bioactive factor mixtures does not lead to a further enhancement in defect healing. Possibly, a synergistic effect can be achieved by further concentration or a prolonged release of bioactive factor mixtures. STATEMENT OF SIGNIFICANCE: : The successful therapy of extended bone defects is still a major challenge in clinical routine. In this study we investigated the bone regenerative potential of naturally occuring bioactive factor mixtures derived from platelet concentrates, adipose tissue and cell secretomes as a cheap and promising alternative to recombinant growth factors in a murine segmental bone defect model. The mixtures alone were not able to induce complete bridging of the bone defect, but in combination with bone morphogenetic protein 2 bone healing seemed to be more physiological. The results show that naturally occuring bioactive factor mixtures are a promising add-on in a clinical setting.

Evaluation of bacterial attachment on mineralized collagen scaffolds and addition of manuka honey to increase mesenchymal stem cell osteogenesis

The design of biomaterials to regenerate bone is likely to increasingly require modifications that reduce bacterial attachment and biofilm formation as infection during wound regeneration can significantly impede tissue repair and typically requires surgical intervention to restart the healing process. Further, much research on infection prevention in bone biomaterials has focused on modeling of non-resorbable metal alloy materials, whereas an expanding direction of bone regeneration has focused on development of bioresorbable materials. This represents a need for the prevention and understanding of infection in resorbable biomaterials. Here, we investigate the ability of a mineralized collagen biomaterial to natively resist infection and examine how the addition of manuka honey, previously identified as an antimicrobial agent, affects gram positive and negative bacterial colonization and mesenchymal stem cell osteogenesis and vasculature formation. We incorporate manuka honey into these scaffolds via either direct fabrication into the scaffold microarchitecture or via soaking the scaffold in a solution of manuka honey after fabrication. Direct incorporation results in a change in the surface characteristics and porosity of mineralized collagen scaffolds. Soaking scaffolds in honey concentrations higher than 10% had significant negative effects on mesenchymal stem cell metabolic activity. Soaking or incorporating 5% honey had no impact on endothelial cell tube formation. Although solutions of 5% honey reduced metabolic activity of mesenchymal stem cells, MSC-seeded scaffolds displayed increased calcium and phosphorous mineral formation, osteoprotegerin release, and alkaline phosphatase activity. Bacteria cultured on mineralized collagen scaffolds demonstrated surfaces covered in bacteria and no method of preventing infection, and using 10 times the minimal inhibitory concentration of antibiotics did not completely kill bacteria within the mineralized collagen scaffolds, indicating bioresorbable scaffold materials may act to shield bacteria from antibiotics. The addition of 5% manuka honey to scaffolds was not sufficient to prevent P. aeruginosa attachment or consistently reduce the activity of methicillin resistant staphylococcus aureus, and concentrations above 7% manuka honey are likely necessary to impact MRSA. Together, our results suggest bioresorbable scaffolds may create an environment conducive to bacterial growth, and potential trade-offs exist for the incorporation of low levels of honey in scaffolds to increase osteogenic potential of osteoprogenitors while high-levels of honey may be sufficient to reduce gram positive or negative bacteria activity but at the cost of reduced osteogenesis.

Amnion and chorion matrix maintain hMSC osteogenic response and enhance immunomodulatory and angiogenic potential in a mineralized collagen scaffold.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

33. Uncoupling of Osteogenic and Osteoclastogenic Differentiation on Nanoparticulate Mineralized Collagen Scaffolds Through Physisorption of Osteoprotegerin

33. Uncoupling of Osteogenic and Osteoclastogenic Differentiation on Nanoparticulate Mineralized Collagen Scaffolds Through Physisorption of Osteoprotegerin

Physical and Chemical Characterization of Biomineralized Collagen with Different Microstructures.

Mineralized collagen is the basic unit in hierarchically organized natural bone with different structures. Polyacrylic acid (PAA) and periodic fluid shear stress (FSS) are the most common chemical and physical means to induce intrafibrillar mineralization. In the present study, non-mineralized collagen, extrafibrillar mineralized (EM) collagen, intrafibrillar mineralized (IM) collagen, and hierarchical intrafibrillar mineralized (HIM) collagen induced by PAA and FSS were prepared, respectively. The physical and chemical properties of these mineralized collagens with different microstructures were systematically investigated afterwards. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that mineralized collagen with different microstructures was prepared successfully. The pore density of the mineralized collagen scaffold is higher under the action of periodic FSS. Fourier transform infrared spectroscopy (FTIR) analysis showed the formation of the hydroxyapatite (HA) crystal. A significant improvement in the pore density, hydrophilicity, enzymatic stability, and thermal stability of the mineralized collagen indicated that the IM collagen under the action of periodic FSS was beneficial for maintaining collagen activity. HIM collagen fibers, which are prepared under the co-action of periodic FSS and sodium tripolyphosphate (TPP), may pave the way for new bone substitute material applications.

Influence of Biomimetically Mineralized Collagen Scaffolds on Bone Cell Proliferation and Immune Activation.

Collagen, as the main component of connective tissue, is frequently used in various tissue engineering applications. In this study, porous sponge-like collagen scaffolds were prepared by freeze-drying and were then mineralized in a simulated body fluid. The mechanical stability was similar in both types of scaffolds, but the mineralized scaffolds (MCS) contained significantly more calcium, magnesium and phosphorus than the unmineralized scaffolds (UCS). Although the MCS contained a lower percentage (~32.5%) of pores suitable for cell ingrowth (113–357 μm in diameter) than the UCS (~70%), the number of human-osteoblast-like MG-63 cells on days 1, 3 and 7 after seeding was higher on MCS than on UCS, and the cells penetrated deeper into the MCS. The cell growth in extracts prepared by eluting the scaffolds for 7 days in a cell culture medium was also markedly higher in the MCS extracts, as indicated by real-time monitoring in the sensory xCELLigence system for 7 days. From this point of view, MCS are more promising for bone tissue engineering than UCS. However, MCS evoked a more pronounced inflammatory response than UCS, as indicated by the production of tumor necrosis factor-alpha (TNF-α) in macrophage-like RAW 264.7 cells in cultures on these scaffolds.

Effect of increasing mineralization on pre-osteoblast response to native collagen fibril scaffolds for bone tissue repair and regeneration.

With limited availability of auto- and allografts, there is increasing demand for alternative bone repair and regeneration materials. Inspired by a mimetic approach, the utility of producing engineered native protein scaffolds is being increasingly realized, demonstrating the need for continued research in this field. In previous work, we detailed a process for producing mineralized collagen scaffolds using tendon to create collagen templates of highly aligned, natively crosslinked collagen fibrils. The process produced mineral phase closely matching that of native bone, and integration of mineral with the collagen template was demonstrated to be easily controlled, allowing scaffolds to be mechanically tuned. In the current study, we have extended this work to investigate how variation in the mineralization level of these scaffolds affects the osteogenic response of pre-osteoblastic cells. Scaffolds were produced under three treatment groups, where collagen templates underwent 0, 5, or 20 mineralization cycles. Scaffolds in each treatment group were cultured with MC3T3-E1 cells for 1, 7, or 14 days. Morphologic assessment under SEM indicated decreased attachment to the mineralized scaffolds, supported by DNA results showing a significant drop between culture days 1 and 7 for mineralized scaffolds only. For adherent cells, increasing scaffold mineralization also delayed cell spreading. While mineralization presented a barrier to cell coverage of scaffolds, it increased osteogenic activity, with cells on the mineralized scaffolds showing significantly greater alkaline phosphatase activity and osteocalcin production. Understanding how increasing collagen mineralization effects pre-osteoblast function may enable design of more advanced mineralized collagen scaffolds for bone repair and regeneration.

Soluble Extracts from Amnion and Chorion Membranes Improve hMSC Osteogenic Response in a Mineralized Collagen Scaffold

Soluble Extracts from Amnion and Chorion Membranes Improve hMSC Osteogenic Response in a Mineralized Collagen Scaffold

Repair of critical-size porcine craniofacial bone defects using a collagen–polycaprolactone composite biomaterial

Regenerative medicine approaches for massive craniomaxillofacial (CMF) bone defects face challenges associated with the scale of missing bone, the need for rapid graft-defect integration, and challenges related to inflammation and infection. Mineralized collagen scaffolds have been shown to promote mesenchymal stem cell osteogenesis due to their porous nature and material properties, but are mechanically weak, limiting surgical practicality. Previously, these scaffolds were combined with 3D-printed polycaprolactone (PCL) mesh to form a scaffold-mesh composite to increase strength and promote bone formation in sub-critical sized porcine ramus defects. Here, we compare the performance of mineralized collagen–PCL composites to the PCL mesh in a critical-sized porcine ramus defect model. While there were no differences in overall healing response between groups, our data demonstrated broadly variable metrics of healing regarding new bone infiltration and fibrous tissue formation. Abscesses were present surrounding some implants and PCL polymer was still present after 9–10 months of implantation. Overall, while there was limited successful healing, with 2 of 22 implants showed substantial levels of bone regeneration, and others demonstrating some form of new bone formation, the results suggest targeted improvements to improve repair of large animal models to more accurately represent CMF bone healing. Notably, strategies to increase osteogenesis throughout the implant, modulate the immune system to support repair, and employ shape-fitting tactics to avoid implant micromotion and resultant fibrosis. Improvements to the mineralized collagen scaffolds involve changes in pore size and shape to increase cell migration and osteogenesis and inclusion or delivery of factors to aid vascular ingrowth and bone regeneration.

Mineralized collagen scaffold bone graft accelerate the osteogenic process of HASCs in proper concentration

PurposeTo investigate the feasibility and the optimum condition of human adipose-derived stem cells cultured on the mineralized collagen material; and to further explore the mechanism of osteogenic differentiation of the human Adipose-derived stem cells stimulated by the mineralized collagen material.MethodsPrimary human adipose-derived stem cells (HADSCs) were isolated from human adipose tissue using centrifugal stratification, which had been passed repeatedly to later generations and purified. Human adipose-derived stem cells were cultured on the bone graft material and the optimum concentration was explored by Alamar blue colorimetric method. The rest experiment was conducted according to the result. The experimental groups are shown below: group A (HADSCs + bone graft material); group B (HADSCs). Morphological observation was taken by scanning electronic microscope (SEM). Alkaline phosphatase activities were tested by histochemical method. Calcium deposition was investigated by alizarin red staining. The quantity access of osteogenic-related mRNA: ALP (alkaline phosphatase), BMP2 (bone morphogenetic protein 2) and RUNX2 (runt-related transcription factor 2) were detected using RT-PCR.ResultsThe cultured cells grew stably and proliferated rapidly. The optimum condition was 0.5 mg/cm2 bone graft material coated on the bottom of medium. After culturing on the material 14 days, the alizarin red staining showed that more calcium deposition was detected in group A and alkaline phosphatase activities of group A was higher than group B (p ˃ 0.05). Similarly, after culturing for 14 days, the ALP, BMP2 and RUNX2 transcription activity of group A was higher than group B (p ˃ 0.05).ConclusionHuman adipose-derived stem cells cultured on bone graft material were dominantly differentiated into osteoblast in vitro. Thus it provided a new choice for bone tissue engineering.

Glycosaminoglycan content of a mineralized collagen scaffold promotes mesenchymal stem cell secretion of factors to modulate angiogenesis and monocyte differentiation

Effective design of biomaterials to aid regenerative repair of craniomaxillofacial (CMF) bone defects requires approaches that modulate the complex interplay between exogenously added progenitor cells and cells in the wound microenvironment, such as osteoblasts, osteoclasts, endothelial cells, and immune cells. We are exploring the role of the glycosaminoglycan (GAG) content in a class of mineralized collagen scaffolds recently shown to promote osteogenesis and healing of craniofacial bone defects. We previously showed that incorporating chondroitin-6-sulfate or heparin improved mineral deposition by seeded human mesenchymal stem cells (hMSCs). Here, we examine the effect of varying scaffold GAG content on hMSC behavior, and their ability to modulate osteoclastogenesis, vasculogenesis, and the immune response. We report the role of hMSC-conditioned media produced in scaffolds containing chondroitin-6-sulfate (CS6), chondroitin-4-sulfate (CS4), or heparin (Heparin) GAGs on endothelial tube formation and monocyte differentiation. Notably, endogenous production by hMSCs within Heparin scaffolds most significantly inhibits osteoclastogenesis via secreted osteoprotegerin (OPG), while the secretome generated by CS6 scaffolds reduced pro-inflammatory immune response and increased endothelial tube formation. All conditioned media down-regulated many pro- and anti-inflammatory cytokines, such as IL6, IL-1β, and CCL18 and CCL17 respectively. Together, these findings demonstrate that modifying mineralized collagen scaffold GAG content can both directly (hMSC activity) and indirectly (production of secreted factors) influence overall osteogenic potential and mineral biosynthesis as well as angiogenic potential and monocyte differentiation towards osteoclastic and macrophage lineages. Scaffold GAG content is therefore a powerful stimulus to modulate reciprocal signaling between multiple cell populations within the bone healing microenvironment.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

Biomimetic mineralized hybrid scaffolds with antimicrobial peptides

Infection in hard tissue regeneration is a clinically-relevant challenge. Development of scaffolds with dual function for promoting bone/dental tissue growth and preventing bacterial infections is a critical need in the field. Here we fabricated hybrid scaffolds by intrafibrillar-mineralization of collagen using a biomimetic process and subsequently coating the scaffold with an antimicrobial designer peptide with cationic and amphipathic properties. The highly hydrophilic mineralized collagen scaffolds provided an ideal substrate to form a dense and stable coating of the antimicrobial peptides. The amount of hydroxyapatite in the mineralized fibers modulated the rheological behavior of the scaffolds with no influence on the amount of recruited peptides and the resulting increase in hydrophobicity. The developed scaffolds were potent by contact killing of Gram-negative Escherichia coli and Gram-positive Streptococcus gordonii as well as cytocompatible to human bone marrow-derived mesenchymal stromal cells. The process of scaffold fabrication is versatile and can be used to control mineral load and/or intrafibrillar-mineralized scaffolds made of other biopolymers.