Direct Osteogenesis Research Articles

Endochondral bone tissue engineering: using cartilage to drive vascularized bone regeneration (87.6)

There is a significant unmet clinical need to develop improved strategies to promote vascularized bone regeneration. Current bone graft technologies promote repair through direct osteogenesis by use of auto‐ or allografted bone. Critical failures associated with these therapies include osteonecrosis and poor osseointegration. The long‐term goal of this project is to develop a tissue engineering approach to promote endochondral bone formation, in which bone forms through a cartilage intermediate. Endochondral ossification is the normal mechanism by which long bones form and the majority of fractures heal. By leveraging this normal healing response, we hypothesize we can overcome the clinical problems of osteonecrosis and poor integration to produce a vascularized and integrated bone regenerate. To test this hypothesis, critical sized defects were created in mid‐diaphysis of murine tibia. Defects remained empty, or were filled with a cartilage graft, autograft or allograft bone. Our data show cartilage grafts effectively transformed into vascularized bone and integrated with the host bone to perform statistically better than allografts and equivalent to autografts. To translate this therapy we have developed a biomimetic acrylated hyaluronic acid hydrogel that contains peptide sequences for cell attachment (bone sialoprotein‐derived RGD), heparin for modulation and sequestration of growth factors, and enzymatically degradable MMP‐13 crosslinks. Hypertrophic chondrocytes encapsulated in these scaffolds mineralization during in vitro culture in basal medium. In ongoing work, these scaffolds have been transplanted into a subcutaneous model of osteonecrosis to evaluate the capacity to re‐vitalize bone. This novel approach to bone tissue engineering has the potential to improve clinical outcomes in bone regeneration.Grant Funding Source: Supported by NIH 5F32AR062469 (CSB), MTF Junior Investigator Award (CSB), UCSF CTSI Award (CSB)

Microbioreactor Array Screening of Wnt Modulators and Microenvironmental Factors in Osteogenic Differentiation of Mesenchymal Progenitor Cells

Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.

Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation.

Although bone has great capacity for repair, there are a number of clinical situations (fracture non-unions, spinal fusions, revision arthroplasty, segmental defects) in which auto- or allografts attempt to augment bone regeneration by promoting osteogenesis. Critical failures associated with current grafting therapies include osteonecrosis and limited integration between graft and host tissue. We speculated that the underlying problem with current bone grafting techniques is that they promote bone regeneration through direct osteogenesis. Here we hypothesized that using cartilage to promote endochondral bone regeneration would leverage normal developmental and repair sequences to produce a well-vascularized regenerate that integrates with the host tissue. In this study, we use a translational murine model of a segmental tibia defect to test the clinical utility of bone regeneration from a cartilage graft. We further test the mechanism by which cartilage promotes bone regeneration using in vivo lineage tracing and in vitro culture experiments. Our data show that cartilage grafts support regeneration of a vascularized and integrated bone tissue in vivo, and subsequently propose a translational tissue engineering platform using chondrogenesis of mesenchymal stem cells (MSCs). Interestingly, lineage tracing experiments show the regenerate was graft derived, suggesting transformation of the chondrocytes into bone. In vitro culture data show that cartilage explants mineralize with the addition of bone morphogenetic protein (BMP) or by exposure to human vascular endothelial cell (HUVEC)-conditioned medium, indicating that endothelial cells directly promote ossification. This study provides preclinical data for endochondral bone repair that has potential to significantly improve patient outcomes in a variety of musculoskeletal diseases and injuries. Further, in contrast to the dogmatic view that hypertrophic chondrocytes undergo apoptosis before bone formation, our data suggest cartilage can transform into bone by activating the pluripotent transcription factor Oct4A. Together these data represent a paradigm shift describing the mechanism of endochondral bone repair and open the door for novel regenerative strategies based on improved biology.

Prednisolone induces osteoporosis-like phenotype in regenerating zebrafish scales

We demonstrate that glucocorticoids induce an osteoporotic phenotype in regenerating scales of zebrafish. Exposure to prednisolone results in altered mineral content, enhanced matrix breakdown, and an osteoporotic gene-expression profile in osteoblasts and osteoclasts. This highlights that the zebrafish scale provides a powerful tool for preclinical osteoporosis research. This study aims to evaluate whether glucocorticoid (prednisolone) treatment of zebrafish induces an osteoporotic phenotype in regenerating scales. Scales, a readily accessible dermal bone tissue, may provide a tool to study direct osteogenesis and its disturbance by glucocorticoids. In adult zebrafish, treated with 25 μM prednisolone phosphate via the water, scales were removed and allowed to regenerate. During regeneration scale morphology and the molar calcium/phosphorus ratio in scales were assessed and osteoblast and osteoclast activities were monitored by time profiling of cell-specific genes; mineralization was visualized by Von Kossa staining, osteoclast activity by tartrate-resistant acid phosphatase histochemistry. Prednisolone (compared to controls) enhances osteoclast activity and matrix resorption and slows down the build up of the calcium/phosphorus molar ratio indicative of altered crystal maturation. Prednisolone treatment further impedes regeneration through a shift in the time profiles of osteoblast and osteoclast genes that commensurates with an osteoporosis-like imbalance in bone formation. A glucocorticoid-induced osteoporosis phenotype as seen in mammals was induced in regenerating scalar bone of zebrafish treated with prednisolone. An unsurpassed convenience and low cost then make the zebrafish scale a superior model for preclinical studies in osteoporosis research.

Local inhibition of 5-lipoxygenase enhances bone formation in a rat model

ObjectivesRecent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis.MethodsBilateral, unicortical femoral defects were used in rats to measure the effects of local 5-LO inhibition on direct osteogenesis. The defect sites were filled with a polycaprolactone (PCL) scaffold containing 5-LO inhibitor (A-79175) at three dose levels, scaffold with drug carrier, or scaffold only. Drug release was assessed in vitro. Osteogenesis was assessed by micro-CT and histology at two endpoints of ten and 30 days.ResultsUsing micro-CT, we found that A-79175, a 5-LO inhibitor, increased bone formation in an apparent dose-related manner.ConclusionsThese results indicate that 5-LO inhibition could be used therapeutically to enhance treatments that require the direct formation of bone.

Tissue engineering bone by recapitulating developmental and repair programs offers improved biological outcomes

The principle behind tissue engineering and regenerative medicine is development of functional replacements for damaged or diseased tissues. In the case of bone, technologies focus on promoting direct osteogenesis. However, current therapies are associated with clinical drawbacks, including osteonecrosis and poor integration. Bone regeneration through endochondral ossification, which is the mechanism by which long bones form and the majority of fractures heal, may overcome these problems. We hypothesize that cartilage grafts can promote a vascularized and integrated bone regenerate. To test this, we created stabilized defects in the mid‐diaphysis of the murine tibia and transplanted cartilage grafts from reporter mice. The cartilage was replaced by vascularized bone that integrated with the host. Since endochondral ossification is thought to require chondrocyte apoptosis and cartilage replacement by osteoblasts from the invading vasculature, we predicted the new bone would be host‐derived. However, the new bone was composed largely of donor‐derived cells and there was limited evidence of apoptosis. Together, these data suggest that in our animal model an alternative healing mechanism, such as transdifferentiation, is occurring. Translation of these data into a patient‐based therapy provides a platform to improve clinical outcomes and gain insight into the biology of bone regeneration.Grant Funding Source: NIH‐NIAMS (R01‐AR053645‐01 to TM), UCSF GEMS‐CSTI Fellowship (CSB)

Chondrogenic pre-induction of human mesenchymal stem cells on β-TCP: Enhanced bone quality by endochondral heterotopic bone formation

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure β-tricalcium-phosphate (β-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. β-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6weeks. After 8weeks, explants were analysed by histology. β-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7mm than with 0.7–1.4mm particles (p⩽0.018). Chondrogenic pre-induction of β-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, β-TCP was significantly more osteo-permissive (p=0.004) than HA/TCP for human MSC, and chondrogenic priming of β-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.

Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone®) osteogenesis: a study on biopsies from human jaws

Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the host's physiological bone turnover at a very early stage.