Mineralized Collagen Glycosaminoglycan Research Articles

Correlating Material Properties to Osteoprotegerin Expression on Nanoparticulate Mineralized Collagen Glycosaminoglycan Scaffolds.

Precision material design directed by cell biological processes represents a frontier in developing clinically translatable regenerative technologies. While understanding cell-material interactions on multipotent progenitor cells yields insights on target tissue differentiation, equally if not more important is the quantification of indirect multicellular interactions. In this work, the relationship of two material properties, phosphate content and stiffness, of a nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG) in the expression of an endogenous anti-osteoclastogenic secreted protein, osteoprotegerin (OPG) by primary human mesenchymal stem cells (hMSCs) is evaluated. The phosphate content of MC-GAG requires the type III sodium phosphate symporter PiT-1/SLC20A1 for OPG expression, correlating with β-catenin downregulation, but is independent of the effects of phosphate ion on osteogenic differentiation. Using three stiffness MC-GAG variants that do not differ significantly by osteogenic differentiation, it is observed that the softest material elicited ≈1.6-2 times higher OPG expression than the stiffer materials. Knockdown of the mechanosensitive signaling axis of YAP, TAZ, β-catenin and combinations thereof in hMSCs on MC-GAG demonstrates that β-catenin downregulation increases OPG expression by 1.5-fold. Taken together, these data constitute a roadmap for material properties that can used to suppress osteoclast activation via osteoprotegerin expression separately from the anabolic processes of osteogenesis.

22. The Role of Orai1 in Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

PURPOSE: Development of synthetic regenerative materials inspired by the extracellular matrix (ECM) has been a topic of significant interest due to the known properties of the ECM to direct cell fate determination via tunable material composition and biomechanical properties. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a low density, open-cell foam that has been evaluated in our laboratory to induce osteogenic differentiation of primary osteoprogenitors in vitro as well as regeneration of rabbit skull defects in vivo without pre-seeding with progenitor cells or addition of exogenous growth factors, which was not observed in non-mineralized collagen glycosaminoglycan (Col-GAG) materials. Given that the critical difference between MC-GAG and Col-GAG is the mineral content, the spatial concentration of inorganic ions on MC-GAG may be critical to its osteogenic properties. This is supported by prior research demonstrating that high calcium concentrations in the bone microenvironment stimulate osteoblastic differentiation. How these biochemical cues are transmitted to cells and activate downstream osteogenic signaling pathways should be further elucidated. Orai1 is a critical subunit of store-operated calcium release-activated calcium channels (CRAC) that has been implicated to be involved in bone homeostasis. In this work, we evaluated the contribution of Orai1 to MC-GAG-mediated osteogenesis. METHODS: To examine the role of extracellular calcium signaling through store-operated calcium channels, such as Orai1, on osteogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs), we treated 2D cell cultures with a small molecule inhibitor of store-operated calcium entry (SOCE), MRS1845, for 14 days. hMSCs were subsequently seeded on MC-GAG or Col-GAG for 7 days with or without MRS1845. Baseline Orai1 gene and protein expression was evaluated. Next, small interfering RNAs (siRNAs) targeting ORAI1 and a scrambled control were used to specifically knockdown ORAI1 expression in hMSCs cultured on Col-GAG and MC-GAG. Osteogenic gene and protein expression were measured using quantitative RT-PCR and western blot analyses, respectively. Mineralization was evaluated by Alizarin Red staining. RESULTS: To understand whether store-operated calcium entry was essential to osteogenic differentiation, we treated hMSCs in 2D cultures with a small molecule inhibitor of SOCE, MRS1845, and found a reduction in calcium depositions visualized with Alizarin Red staining when compared to untreated hMSCs. In 3D cultures, MRS1845 downregulated the gene expression of early osteogenic marker ALP and protein expression of the intracellular osteogenic mediator Runx2 specifically on MC-GAG compared to controls. As small molecule inhibitors lack channel specificity, we knocked down ORAI1 expression through siRNA transfection. When MC-GAG was compared with Col-GAG, baseline ORAI1 gene expression was 1.8-fold higher (p=0.02) and Orai1 protein expression was upregulated (p=0.003). ORAI1 knockdown reduced the protein expression of Runx2 and gene expression of late osteogenic marker BSP2 specifically on MC-GAG compared to scrambled control siRNA. Mineralization significantly diminished on MC-GAG with ORAI1 knockdown, whereas no effects were evident on Col-GAG as demonstrated by Alizarin Red staining. CONCLUSIONS: ORAI1-mediated calcium ion signaling was required for MC-GAG-induced osteogenic differentiation. Further elucidation of the osteogenic mechanisms and refinement of calcium levels on MC-GAG may facilitate its clinical application in the reconstruction of osseous defects.

Modulating Temporospatial Phosphate Equilibrium by Nanoparticulate Mineralized Collagen Materials Induces Osteogenesis via PiT-1 and PiT-2.

The temporospatial equilibrium of phosphate contributes to physiological bone development and fracture healing, yet optimal control of phosphate content has not been explored in skeletal regenerative materials. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a synthetic, tunable material that promotes in vivo skull regeneration. In this work, the effects of MC-GAG phosphate content on the surrounding microenvironment and osteoprogenitor differentiation are investigated. This study finds that MC-GAG exhibits a temporal relationship with soluble phosphate with elution early in culture shifting to absorption with or without differentiating primary bone marrow-derived human mesenchymal stem cells (hMSCs). The intrinsic phosphate content of MC-GAG is sufficient to stimulate osteogenic differentiation of hMSCs in basal growth media without the addition of exogenous phosphate in a manner that can be severely reduced, but not eliminated, by knockdown of the sodium phosphate transporters PiT-1 or PiT-2. The contributions of PiT-1 and PiT-2 to MC-GAG-mediated osteogenesis are nonredundant but also nonadditive, suggestive that the heterodimeric form is essential to its activity. These findings indicate that the mineral content of MC-GAG alters phosphate concentrations within a local microenvironment resulting in osteogenic differentiation of progenitor cells via both PiT-1 and PiT-2.

Osteoprotegerin-eluting nanoparticulate mineralized collagen scaffolds improve skull regeneration

Custom synthesis of extracellular matrix (ECM)-inspired materials for condition-specific reconstruction has emerged as a potentially translatable regenerative strategy. In skull defect reconstruction, nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) have demonstrated osteogenic and anti-osteoclastogenic properties, culminating in the ability to partially heal in vivo skull defects without the addition of exogenous growth factors or progenitor cell loading. In an effort to reduce catabolism during early skull regeneration, we fabricated a composite material (MCGO) of MC-GAG and recombinant osteoprotegerin (OPG), an endogenous anti-osteoclastogenic decoy receptor. In the presence of differentiating osteoprogenitors, MCGO demonstrated an additive effect with endogenous OPG limited to the first 14 days of culture with total eluted and scaffold-bound OPG exceeding that of MC-GAG. Functionally, MCGO exhibited similar osteogenic properties as MC-GAG, however, MCGO significantly reduced maturation and resorptive activities of primary human osteoclasts. In a rabbit skull defect model, MCGO scaffold-reconstructed defects displayed higher mineralization as well as increased hardness and microfracture resistance compared to non-OPG functionalized MC-GAG scaffolds. The current work suggests that MCGO is a development in the goal of reaching a materials-based strategy for skull regeneration.

Inhibition of the Wnt Pathway Reduces Activation of the YAP/TAZ Mechanotransduction Pathways and Osteogenic Differentiation in Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

PURPOSE: Developing surgically practical regenerative materials requires in-depth understanding of cell-material interaction mechanisms. We previously identified a novel nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material—inspired by the extracellular matrix—with the ability to induce calvarial bone healing without added exogenous growth factors or progenitor cells, suggesting that this material may support the development of off-the-shelf implants for skull defects. We have demonstrated that modulating the material’s stiffness activates the mechanotransduction pathway via YAP and TAZ, thus improving osteogenic differentiation. Given the strong relationship between the mechanotransduction pathways and Wnt activation, this work seeks to determine the necessity of the Wnt pathways for the activity of MC-GAG in relationship to differences in stiffness. METHODS: MC-GAG scaffolds were prepared with lyophilization. Crosslinking was performed with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 5:2:1 EDC:NHS:COOH; COOH represents the amount of collagen in the scaffold. Noncrosslinked (NX-MC) and crosslinked (MC) GAG scaffolds were cultured with primary bone-marrow-derived human mesenchymal stem cells. Osteogenic differentiation was assessed with a combination of quantitative real-time reverse transcriptase polymerase chain reaction for osteogenic differentiation markers, western blot and confocal microscopy for activation and subcellular localization of intracellular mediators, and alizarin red staining and micro-computed tomography for mineralization. Wnt pathways were inhibited using small molecular inhibitors IWR1 (specifically targeting the canonical Wnt/b-catenin pathway) and IWP2 (targeting both canonical and non-canonical Wnt pathways). Analyses of variance and Tukey post hoc testing were performed with a P < 0.05 significance threshold. RESULTS: Inhibiting the Wnt pathways with IWR1 and IWP2 resulted in decreased mineralization of primary human mesenchymal stem cells on MC scaffolds, whereas NX-MC scaffolds were unaffected. Both inhibitors downregulated, but did not eliminate, gene expression of bone markers, including alkaline phosphatase, collagen 1, and bone sialoprotein II primarily affecting MC over NX-MC. However, a reciprocal increase in BMP4 expression was found in the presence of both inhibitors. On western blot analysis, both inhibitors reduced protein expression of active b-catenin, YAP, and TAZ on MC materials, whereas the less stiff NX-MC material demonstrated no differences in expression. Despite the reduction in bone markers and mineralization, a reciprocal increase in Runx2 and Smad1/5 phosphorylation was noted in the presence of IWR1 and IWP2 specifically on the MC scaffolds, but not on NX-MC. On confocal microscopy, untreated materials exhibited colocalization of YAP and active b-catenin in the cytosolic compartment on NX-MC and in both cytosolic and nuclear compartments on MC. While IWR1 and IWP2 did not affect YAP and active b-catenin staining on NX-MC scaffolds, they reduced nuclear localization of active b-catenin on MC scaffolds, with IWP2 demonstrating more of an effect than IWR1. CONCLUSIONS: Mechanistic understanding of regenerative materials is required for safe clinical translation. We have shown that stiffness of MC-GAG, a promising skull regenerative material, can improve osteogenic capabilities via YAP/TAZ-mediated mechanotransduction. In this work, we showed that the osteogenic properties, imparted by the stiffness of MC-GAG, function through the Wnt signaling pathways, such that inhibition downregulates YAP/TAZ expression, mineralization, and expression of bone markers.

β-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner.

Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is shown to promote skull regeneration in vivo without additive exogenous growth factors or progenitor cells, suggesting potential for clinical translation. This work evaluates modulation of MC-GAG stiffness on canonical Wnt (cWnt) signaling. Primary human bone marrow-derived mesenchymal stem cells (hMSCs) are differentiated on two MC-GAG scaffolds (noncrosslinked, NX-MC, 0.3kPa vs conventionally crosslinked, MC, 3.9kPa). hMSCs increase expression of activated β-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization on stiffer MC scaffolds. Overall Wnt pathway inhibition reduces activated β-catenin and osteogenic differentiation, while elevating BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike Wnt pathway downregulation, isolated canonical Wnt inhibition with β-catenin knockdown increases osteogenic differentiation and mineralization specifically on the stiffer MC. β-catenin knockdown also increases p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Thus, while stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis on MC-GAG, activated β-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials.

Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling.

The ability of the extracellular matrix (ECM) to instruct progenitor cell differentiation has generated excitement for the development of materials-based regenerative solutions. Described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material capable of inducing in vivo skull regeneration without exogenous growth factors or ex vivo progenitor cell-priming is described previously. Here, the contribution of titrating stiffness to osteogenicity is evaluated by comparing noncrosslinked (NX-MC) and crosslinked (MC) forms of MC-GAG. While both materials are osteogenic, MC demonstrates an increased expression of osteogenic markers and mineralization compared to NX-MC. Both materials are capable of autogenously activating the canonical BMPR signaling pathway with phosphorylation of Smad1/5. However, unlike NX-MC, human mesenchymal stem cells cultured on MC demonstrate significant elevations in the major mechanotransduction mediators YAP and TAZ expression, coincident with β-catenin activation in the canonical Wnt signaling pathway. Inhibition of YAP/TAZ activation reduces osteogenic expression, mineralization, and β-catenin activation in MC, with less of an effect on NX-MC. YAP/TAZ inhibition also results in a reciprocal increase in Smad1/5 phosphorylation and BMP2 expression. The results indicate that increasing MC-GAG stiffness induces osteogenic differentiation via the mechanotransduction mediators YAP/TAZ and the canonical Wnt signaling pathway, whereas the canonical BMPR signaling pathway is activated independent of stiffness.

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.

Cover Image, Volume 13, Issue 5

The cover image is based on the Research Article Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation by Xiaoyan Ren et al., https://doi.org/10.1002/term.2834.

Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation.

The ability of the extracellular matrix (ECM) to direct cell fate has generated the potential for developing a materials-only strategy for tissue regeneration. Previously, we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion. In this work, we evaluated the interactions between MC-GAG and primary human osteoclasts (hOCs). In the absence of hMSCs, mineralized Col-GAG materials directly inhibited hOC viability, proliferation, and resorption in contrast to nonmineralized Col-GAG, which demonstrated a modest inhibition of resorptive activity only. Cocultures containing differentiating hMSCs with hOCs demonstrated increased hOC-mediated resorption only on Col-GAG while MC-GAG cocultures continued to inhibit resorption. Unlike Col-GAG, hMSCs on MC-GAG expressed increased amounts of osteoprotegerin (OPG) protein, the major endogenous osteoclast inhibitor. Interestingly, OPG expression was found to be antagonized by small mothers against decapentaplegic1/5 (Smad1/5) phosphorylation, an obligate pathway for osteogenic differentiation of hMSCs on MC-GAG, and potentiated by extracellular signal-regulated kinase (ERK1/2) phosphorylation. Collectively, these results suggested that the MC-GAG material both directly inhibited the osteoclast viability, proliferation, and resorptive activity as well as induced hMSCs to secrete osteoprotegerin, an antiosteoclastogenic factor, via a signalling pathway distinct from osteogenic differentiation.

Abstract 90: L-Type Voltage Gated Calcium Channels and Sodium Phosphate Symporters are Required for Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Materials

PURPOSE: The increasing understanding that modulation of extracellular matrix (ECM) components can instruct differential cell fate determination has resulted in significant interest in the synthesis of ECM-inspired materials for regeneration.Previously we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion via an autogenous activation of the bone morphogenetic protein receptor (BMPR) signaling pathway. Such activities were not induced in non-mineralized collagen glycosaminoglycan (Col-GAG) materials, suggesting that the upstream trigger is related to the inorganic content of the material. In this work, we evaluated the contributions of inorganic ion signaling to MC-GAG-induced osteogenic differentiation. METHODS: Primary hMSCs were osteogenically differentiated on collagen glycosaminoglycan materials in the absence or presence of inhibitors to the L-type voltage gated calcium channels (L-VGCC), calcium sensing receptor (CaSR), and the sodium phosphate symporters (PiT-1/2) using nifedipine, NPS2143,and phosphonoformic acid (PFA), respectively. Western blots were performed for phosphorylated and total Smad1/5, ERK1/2, Akt, p38, JNK1/2, total Runx2, and actin. Expression of BMPs were assessed with immunofluorescent staining of scaffold sections. Mineralization was evaluated by Alizarin red staining and micro-computed tomographic analyses. Analyses of variance with posthoc comparisons were performed. RESULTS: PFA diminished phosphorylated Smad1/5, BMP-2, and Runx2 expression as well as mineralization for both Col-GAG and MC-GAG scaffolds. While NPS2143 diminished Runx2 and mineralization on Col-GAG, no effects on osteogenic protein expression, BMP-2 expression, or mineralization were evident on MC-GAG. In contrast, no effects were mediated by nifedipine on Col-GAG,whereas nifedipine inhibited phosphorylated Smad1/5, phosphorylated ERK1/2, Runx2, BMP-2 staining, and mineralization on MC-GAG. CONCLUSION: In combination, these data suggested that the autogenous activation of the BMPR signaling pathway induced by MC-GAGis first triggered by calcium and phosphate influxvia L-VGCCs and the sodium and phosphate symporters.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Abstract: Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells on Collagen Glycosaminoglycan Scaffolds

Non-Mineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells INTRODUCTION: The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, we described a correlation between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of Smad1/5 in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished ERK1/2 activation when compared to non-mineralized scaffolds (Col-GAG). In this work, we utilize a canonical BMPR inhibitor, DMH1, and an inhibitor of the MEK/ERK cascade, PD98059, to characterize the necessity of each pathway for osteogenesis. METHODS: hMSCs were cultured in osteogenic media on Col-GAG or MC-GAG scaffolds. Scaffolds were untreated or treated with the DMH1 or PD98059 at 50 mM for 4 days, 14 days, 24 days, 4 weeks, and 8 weeks. Gene and protein expression were measured using quantitative RT-PCR and western blot analysis. Scaffolds were subjected to histochemical and micro-computed tomographic analyses. RESULTS: Inhibition of the BMPR signaling pathway inhibited Runx2 and BSPII gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG. In contrast, inhibition of the MEK/ERK axis downregulated BSPII expression on Col-GAG independent of Runx2 expression. While inhibition of the BMPR signaling pathway resulted in decreased mineralization on both Col-GAG and MC-GAG, inhibition of the MEK/ERK axis only affected mineralization on Col-GAG. When the mechanistic detais were evaluated in greater detail, inhibition of the BMPR pathway reduced both Smad1/5 phosphorylation and Runx2 protein expression on both MC-GAG and Col-GAG. Inhibition of the MEK/ERK axis downregulated phosphorylation of ERK1/2 and JNK1/2 without affecting Smad1/5 phosphorylation or Runx2 expression. CONCLUSION: Interactions between hMSCs and collagen-based materials result in mechanistic differences in osteogenesis. Activation of the canonical BMPR signaling is required for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Abstract 137: Contribution of the Non-Canonical BMP Receptor Signaling Pathway Osteogenesis on Bioinspired Nanoparticulate Mineralized Collagen Scaffolds

PURPOSE: We recently described a bioinspired material composed of nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) that induces efficient osteogenic differentiation of human mesenchymal stem cells (hMSCs) without the addition of exogenous growth factors, thereby suggestive of a promising, growth factor-free, materials-based method for clinically utilizable bone regeneration. We have previously reported that MC-GAG scaffolds autogenously induce BMP receptor signaling via the canonical (Smad1/5) pathway. However, the necessary and sufficient components of this mechanism remain unknown. Our current study evaluates the contribution of the non-canonical (MAP kinase) pathway in MC-GAG induced osteogenesis. METHODS: hMSCs were cultured in osteogenic media on non-mineralized (Col-GAG) and nanoparticulate mineralized (MC-GAG) collagen glycosaminoglycan scaffolds. Scaffolds were untreated or treated with the MAP kinase inhibitor PD98059 at 20 μM and 50 μM for 4 days, 14 days, 24 days, 4 weeks, and 8 weeks. Gene and protein expression were measured using quantitative RT-PCR and western blot analysis. Scaffolds were subjected to histochemical and micro-computed tomographic analyses. RESULTS: Inhibition of the non-canonical BMP receptor signaling pathway resulted in a decrease in expression of early osteogenic genes. Western blot analysis demonstrated that PD98059 inhibited phosphorylation of ERK1/2 and JNK without affecting Smad1/5, p38, and Akt phosphorylation. At 4 and 8 weeks of culture, Runx2 protein expression and mineralization on μCT scans were decreased in the presence of PD98059. CONCLUSION: Activation of the non-canonical BMP receptor signaling pathway is essential for osteogenic differentiation of hMSCs on MC-GAG scaffolds.

Evaluation of multi-scale mineralized collagen–polycaprolactone composites for bone tissue engineering

A particular challenge in biomaterial development for treating orthopedic injuries stems from the need to balance bioactive design criteria with the mechanical and geometric constraints governed by the physiological wound environment. Such trade-offs are of particular importance in large craniofacial bone defects which arise from both acute trauma and chronic conditions. Ongoing efforts in our laboratory have demonstrated a mineralized collagen biomaterial that can promote human mesenchymal stem cell osteogenesis in the absence of osteogenic media but that possesses suboptimal mechanical properties in regards to use in loaded wound sites. Here we demonstrate a multi-scale composite consisting of a highly bioactive mineralized collagen–glycosaminoglycan scaffold with micron-scale porosity and a polycaprolactone support frame (PCL) with millimeter-scale porosity. Fabrication of the composite was performed by impregnating the PCL support frame with the mineral scaffold precursor suspension prior to lyophilization. Here we evaluate the mechanical properties, permeability, and bioactivity of the resulting composite. Results indicated that the PCL support frame dominates the bulk mechanical response of the composite resulting in a 6000-fold increase in modulus compared to the mineral scaffold alone. Similarly, the incorporation of the mineral scaffold matrix into the composite resulted in a higher specific surface area compared to the PCL frame alone. The increased specific surface area in the collagen-PCL composite promoted increased initial attachment of porcine adipose derived stem cells versus the PCL construct.

Nanoparticulate mineralized collagen scaffolds induce in vivo bone regeneration independent of progenitor cell loading or exogenous growth factor stimulation

Current strategies for skeletal regeneration often require co-delivery of scaffold technologies, growth factors, and cellular material. However, isolation and expansion of stem cells can be time consuming, costly, and requires an additional procedure for harvest. Further, the introduction of supraphysiologic doses of growth factors may result in untoward clinical side effects, warranting pursuit of alternative methods for stimulating osteogenesis. In this work, we describe a nanoparticulate mineralized collagen glycosaminoglycan scaffold that induces healing of critical-sized rabbit cranial defects without addition of expanded stem cells or exogenous growth factors. We demonstrate that the mechanism of osteogenic induction corresponds to an increase in canonical BMP receptor signalling secondary to autogenous production of BMP-2 and -9 early and BMP-4 later during differentiation. Thus, nanoparticulate mineralized collagen glycosaminoglycan scaffolds may provide a novel growth factor-free and ex vivo progenitor cell culture-free implantable method for bone regeneration.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Density–property relationships in collagen–glycosaminoglycan scaffolds

Collagen–glycosaminoglycan scaffolds for the regeneration of skin have previously been fabricated by freeze-drying a slurry containing a co-precipitate of collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry compressive Young’s modulus is roughly 30 kPa and the dry compressive strength is roughly 5 kPa). There is interest in using these scaffolds for tendon and ligament regeneration where there is a need for improved mechanical properties. Previous attempts to increase the mechanical properties of the scaffold by increasing the solid volume fraction of the scaffolds were limited by the increasing viscosity of the slurry, making it more difficult to mix and giving inhomogeneous scaffolds. Our recent work on mineralized collagen–glycosaminoglycan scaffolds used a vacuum filtration technique to increase the volume fraction of solids in the slurry, thereby increasing the density and mechanical properties of the scaffolds. In this work, we used this technique to fabricate collagen–glycosaminoglycan scaffolds with dry densities between 0.0076 and 0.0311 g cm −3 and pore sizes between 250 and 350 μm, values appropriate for soft tissue growth. The compressive Young’s modulus and strength in the dry state increased from 32 to 127 kPa and from 5 to 19 kPa, respectively, with increasing density. The tensile Young’s modulus in the dry state increased from 295 to 3.1 MPa with increasing density. Finally, we showed that the attachment of cells onto the scaffold was directly proportional to the specific surface area of the scaffold, which defines the total internal surface area per volume of scaffold.

Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen–glycosaminoglycan scaffold

This paper is the second in a series of papers describing the design and development of an osteochondral scaffold using collagen-glycosaminoglycan and calcium phosphate technologies engineered for the regenerative repair of articular cartilage defects. The previous paper described a technology (concurrent mapping) for systematic variation and control of the chemical composition of triple coprecipitated collagen, glycosaminoglycan, and calcium phosphate (CGCaP) nanocomposites without using titrants. This paper describes (1) fabricating porous, three-dimensional scaffolds from the CGCaP suspensions, (2) characterizing the microstructure and mechanical properties of such scaffolds, and (3) modifying the calcium phosphate mineral phase. The methods build on the previously demonstrated ability to vary the composition of a CGCaP suspension (calcium phosphate mass fraction between 0 and 80 wt %) and enable the production of scaffolds whose pore architecture (mean pore size: 50-1000 microm), CaP phase chemistry (brushite, octacalcium phosphate, apatite) and crosslinking density (therefore mechanical properties and degradation rate) can be independently controlled. The scaffolds described in this paper combine the desirable biochemical properties and pore architecture of porous collagen-glycosaminoglycan scaffolds with the strength and direct bone-bonding properties of calcium phosphate biomaterials in a manner that can be tailored to meet the demands of a range of applications in orthopedics and regenerative medicine.