Engineering of an Osteoinductive and Growth Factor-Free Injectable Bone-Like Microgel for Bone Regeneration.

Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein-loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor-free bone-like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro-constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical-sized defects in-vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.

Amniotic membrane attenuates heterotopic ossification following high-dose bone morphogenetic protein-2 treatment of segmental bone defects.

Treatment of large bone defects with supraphysiological doses of bone morphogenetic protein-2 (BMP-2) has been associated with complications including heterotopic ossification (HO), inflammation, and pain, presumably due to poor spatiotemporal control of BMP-2. We have previously recapitulated extensive HO in our rat femoral segmental defect model by treatment with high-dose BMP-2 (30 µg). Using this model and BMP-2 dose, our objective was to evaluate the utility of a clinically available human amniotic membrane (AM) around the defect space for guided bone regeneration and reduction of HO. We hypothesized that AM surrounding collagen sponge would attenuate heterotopic ossification compared with collagen sponge alone. In vitro, AM retained more BMP-2 than a synthetic poly(ε-caprolactone) membrane through 21 days. In vivo, as hypothesized, the collagen + AM resulted in significantly less heterotopic ossification and correspondingly, lower total bone volume (BV), compared with collagen sponge alone. Although bone formation within the defect was delayed with AM around the defect, by 12 weeks, defect BVs were equivalent. Torsional stiffness was significantly reduced with AM but was equivalent to that of intact bone. Collagen + AM resulted in the formation of dense fibrous tissue and mineralized tissue, while the collagen group contained primarily mineralized tissue surrounded by marrow-like structures. Especially in conjunction with high doses of growth factor delivered via collagen sponge, these findings suggest AM may be effective as an overlay adjacent to bone healing sites to spatially direct bone regeneration and minimize heterotopic ossification.

Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss.

Traumatic composite bone-muscle injuries, such as open fractures, often require multiple surgical interventions and still typically result in long-term disability. Clinically, a critical indicator of composite injury severity is vascular integrity; vascular damage alone is sufficient to assign an open fracture to the most severe category. Challenging bone injuries are often treated with bone morphogenetic protein 2 (BMP-2), an osteoinductive growth factor, delivered on collagen sponge. Previous studies in a composite defect model found that a minimally bridging dose in the segmental defect model was unable to overcome concomitant muscle damage, but the effect of BMP dose on composite injuries has not yet been studied. Here, we test the hypotheses that BMP-2-mediated functional regeneration of composite extremity injuries is dose dependent and can be further enhanced via co-delivery of adipose-derived microvascular fragments (MVF), which have been previously shown to increase tissue vascular volume. Although MVF did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. This is the first known report of an increased BMP-2 dose improving bone healing with concomitant muscle damage. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 µg dose used in this study did not result in HO and was associated with a lower circulating inflammatory cytokine profile than the low dose (2.5 µg) group. These data support the potential benefits of an increased, though still moderate, BMP-2 dose for treatment of bone defects with concomitant muscle damage. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res.

Delivery vehicle effects on bone regeneration and heterotopic ossification induced by high dose BMP-2.

Bone morphogenetic protein-2 (BMP-2), delivered on absorbable collagen sponge, is frequently used to treat bone defects. However, supraphysiological BMP-2 doses are common and often associated with complications such as heterotopic ossification and inflammation, causing pain and impaired mobility. This has prompted investigations into strategies to spatially control bone regeneration, for example growth factor delivery in appropriate scaffolds. Our objective was to investigate the spatiotemporal effects of high dose BMP-2 on bone regeneration as a function of the delivery vehicle. We hypothesized that an alginate delivery system would spatially restrict bone formation compared to a collagen sponge delivery system. In vitro, BMP-2 release was accelerated from collagen sponge compared to alginate constructs. In vivo, bone regeneration was evaluated over 12weeks in critically sized rat femoral segmental defects treated with 30µg rhBMP-2 in alginate hydrogel or collagen sponge, surrounded by perforated nanofiber meshes. Total bone volume, calculated from micro-CT reconstructions, was higher in the alginate group at 12weeks. Though bone volume within the central defect region was greater in the alginate group at 8 and 12weeks, heterotopic bone volume was similar between groups. Likewise, mechanical properties from ex vivo torsional testing were comparable between groups. Histology corroborated these findings and revealed heterotopic mineralization at 2weeks post-surgery in both groups. Overall, this study recapitulated the heterotopic ossification associated with high dose BMP-2 delivery, and demonstrated that the amount and spatial pattern of bone formation was dependent on the delivery matrix. STATEMENT OF SIGNIFICANCE: Alginate hydrogel-based BMP-2 delivery has induced better spatiotemporal bone regeneration in animals, compared to clinically used collagen sponge, at lower BMP-2 doses. Lack of clear dose-response relationships for BMP-2 vis-à-vis bone regeneration has contributed to the use of higher doses clinically. We investigated the potential of the alginate system, with comparatively favorable BMP-2 release-kinetics, to reduce heterotopic ossification and promote bone regeneration, when used with a high BMP-2 dose. While defect mineralization improved with alginate hydrogel, the initial high-release phase and likely early tissue exposure to BMP-2 appeared sufficient to induce heterotopic ossification. The characterization presented here should provide the framework for future evaluations of strategies to optimize bone formation and minimize adverse effects of high dose BMP-2 therapy.

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template.

In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and ß-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31+, vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31+) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair.

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the alpha(2)beta(1) integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

Noninvasive image analysis of 3D construct mineralization in a perfusion bioreactor.

Although the beneficial effects of perfusion on cell-mediated mineralization have been demonstrated in several studies, the size of the mineralized constructs produced has been limited. The ability to quantify mineralized matrix formation non-invasively within 3D constructs would benefit efforts to optimize bioreactor conditions for scaling-up constructs to clinically relevant dimensions. In this study, we report a micro-CT imaging-based technique to monitor 3D mineralization over time in a perfusion bioreactor and specifically assess mechanisms of construct mineralization by quantifying the number, size, and distribution of mineralized particle formation within constructs varying in thickness from 3 to 9 mm. As expected, mineralized matrix volume and particle number increased with construct thickness. Analyzing multiple concentric volumes inside each construct indicated that a greater proportion of the mineral volume was found within the interior of the perfused constructs. Interestingly, intermediate-sized 6mm thick constructs were found to have the highest core mineral volume fraction and the largest mineralized particles. Two complementary mechanisms of increasing total mineral volume were observed in the 6 and 9 mm constructs: increasing particle size and increasing the number of mineralized particles, respectively. The rate of mineralized matrix formation in the perfused constructs increased from 0.69 mm(3)/week during the first 3 weeks of culture to 1.03 mm(3)/week over the final 2 weeks. In contrast, the rate of mineral deposition in the static controls was 0.01 mm(3)/week during the first 3 weeks of culture and 0.16 mm(3)/week from week 3 to week 5. The ability to monitor overall construct mineralization non-invasively coupled with quantitative analysis of mineralized particle size, number, and distribution offers a powerful tool for elucidating how mineral growth mechanisms are affected by cell type, scaffold material and architecture, or bioreactor flow conditions.

Microcomputed tomography imaging of skeletal development and growth.

Skeletogenesis is an exquisitely orchestrated and dynamic process, culminating in the formation of highly variable and complex mineralized structures that are optimized for their function. While cellular and molecular biology studies have provided tremendous recent progress toward understanding how patterns of bone formation are regulated, high resolution imaging techniques such as microcomputed tomography (micro-CT) can provide complementary quantitative information about the progressive changes in three-dimensional (3-D) skeletal morphology and density that occur during early skeletal development and postnatal growth. Furthermore, recently developed in vivo micro-CT systems promise to be a powerful and efficient tool for noninvasively monitoring normal skeletogenesis, as well as for evaluating the effects of genetic or environmental manipulation. This review focuses on the use of micro-CT imaging and analysis to better understand normal and abnormal skeletal development and growth.