Mineralized collagen scaffolds for regenerative engineering applications.

Collagen is a primary constituent of the tissue extracellular matrix. As a result, collagen has been a common component of tissue engineering biomaterials, including those to promote bone regeneration or to investigate cell-material interactions in the context of bone homeostasis or disease. This review summarizes key considerations regarding current state-of-the-art design and use of collagen biomaterials for these applications. We also describe strategic opportunities for collagen biomaterials to address a new era of challenges, including immunomodulation and appropriate consideration of sex and other patient characteristics in biomaterial design.

DONOR VARIABILITY IN HUMAN MESENCHYMAL STEM CELL OSTEOGENIC RESPONSE AS A FUNCTION OF PASSAGE CONDITIONS AND DONOR SEX.

Contemporary tissue engineering efforts often seek to use mesenchymal stem cells (MSCs) due to their potential to differentiate to various tissue-specific cells and generate a pro-regenerative secretome. While MSC differentiation and therapeutic potential can differ as a function of matrix environment, it may also be widely influenced as a function of donor-to-donor variability. Further, effects of passage number and donor sex may further convolute the identification of clinically effective MSC-mediated regeneration technologies. We report efforts to adapt a well-defined mineralized collagen scaffold platform to study the influence of MSC proliferation and osteogenic potential as a function of passage number and donor sex. Mineralized collagen scaffolds broadly support MSC osteogenic differentiation and regenerative potency in the absence of traditional osteogenic supplements for a wide range of MSCs (rabbit, rat, porcine, human). We obtained a library of bone marrow and adipose tissue derived stem cells to examine donor-variability of regenerative potency in mineralized collagen scaffolds. MSCs displayed reduced proliferative capacity as a function of passage duration. Further, MSCs showed significant sex-based differences. Notably, MSCs from male donors displayed significantly higher metabolic activity and proliferation while MSCs from female donor displayed significantly higher osteogenic response via increased alkaline phosphate activity, osteoprotegerin release, and mineral formation in vitro. Our study highlights the essentiality of considering MSC donor sex and culture expansion in future studies of biomaterial regenerative potential.

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.

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.

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.

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.

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.

Correction: Co-self-assembly of multiple DNA origami nanostructures in a single pot.

Correction for 'Co-self-assembly of multiple DNA origami nanostructures in a single pot' by Joshua A. Johnson et al., Chem. Commun., 2021, 57, 4795-4798, DOI: 10.1039/D1CC00049G.

ß-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, 0.3 kPa vs conventionally crosslinked, MC, 3.9 kPa). hMSCs increase expression of activated ß-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 ß-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 ß-catenin knockdown increases osteogenic differentiation and mineralization specifically on the stiffer MC. ß-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 ß-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials.

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-1ß, 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.

Co-self-assembly of multiple DNA origami nanostructures in a single pot.

Simultaneous self-assembly of two distinct DNA origami nanostructures folded with the same scaffold strand was achieved in a single pot. Relative yields were tuned by adjusting concentrations of the competing strands, correlating well with folding kinetics of individual structures. These results can faciliate efficient fabrication of multi-structure systems and materials.