Extracellular matrix compression temporally regulates microvascular angiogenesis.

Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis.

OBJECTIVE: To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. DESIGN: Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. RESULTS: Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. CONCLUSION: These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

Contrast enhanced µCT imaging of early articular changes in a pre-clinical model of osteoarthritis.

OBJECTIVE: The objective of this study was to characterize early osteoarthritis (OA) development in cartilage and bone tissues in the rat medial meniscus transection (MMT) model using non-destructive equilibrium partitioning of an ionic contrast agent micro-computed tomography (EPIC-µCT) imaging. Cartilage fibrillation, one of the first physiological developments in OA, was quantified in the rat tibial plateau as three-dimensional (3D) cartilage surface roughness using a custom surface-rendering algorithm. METHODS: Male Lewis rats underwent MMT or sham-operation in the left leg. At 1- and 3-weeks post-surgery, the animals (n = 7-8 per group) were euthanized and the left legs were scanned using EPIC-µCT imaging to quantify cartilage and bone parameters. In addition, a custom algorithm was developed to measure the roughness of 3D surfaces. This algorithm was validated and used to quantify cartilage surface roughness changes as a function of time post-surgery. RESULTS: MMT surgery resulted in significantly greater cartilage damage and subchondral bone sclerosis with the damage increasing in both severity and area from 1- to 3-weeks post-surgery. Analysis of rendered 3D surfaces could accurately distinguish early changes in joints developing OA, detecting significant increases of 45% and 124% in surface roughness at 1- and 3-weeks post-surgery respectively. CONCLUSION: Disease progression in the MMT model progresses sequentially through changes in the cartilage articular surface, extracellular matrix composition, and then osteophyte mineralization and subchondral bone sclerosis. Cartilage surface roughness is a quantitative, early indicator of degenerative joint disease in small animal OA models and can potentially be used to evaluate therapeutic strategies.

Quantitative pre-clinical screening of therapeutics for joint diseases using contrast enhanced micro-computed tomography.

OBJECTIVE: The development of effective therapies for cartilage protection has been limited by a lack of efficient quantitative cartilage imaging modalities in pre-clinical in vivo models. Our objectives were two-fold: first, to validate a new contrast-enhanced 3D imaging analysis technique, equilibrium partitioning of an ionic contrast agent-micro computed tomography (EPIC-µCT), in a rat medial meniscal transection (MMT) osteoarthritis (OA) model; and second, to quantitatively assess the sensitivity of EPIC-µCT to detect the effects of matrix metalloproteinase inhibitor (MMPi) therapy on cartilage degeneration. METHODS: Rats underwent MMT surgery and tissues were harvested at 1, 2, and 3 weeks post-surgery or rats received an MMPi or vehicle treatment and tissues harvested 3 weeks post-surgery. Parameters of disease progression were evaluated using histopathology and EPIC-µCT. Correlations and power analyses were performed to compare the techniques. RESULTS: EPIC-µCT was shown to provide simultaneous 3D quantification of multiple parameters, including cartilage degeneration and osteophyte formation. In MMT animals treated with MMPi, OA progression was attenuated, as measured by 3D parameters such as lesion volume and osteophyte size. A post-hoc power analysis showed that 3D parameters for EPIC-µCT were more sensitive than 2D parameters requiring fewer animals to detect a therapeutic effect of MMPi. 2D parameters were comparable between EPIC-µCT and histopathology. CONCLUSION: This study demonstrated that EPIC-µCT has high sensitivity to provide 3D structural and compositional measurements of cartilage and bone in the joint. EPIC-µCT can be used in combination with histology to provide a comprehensive analysis to screen new potential therapies.

Biomaterial strategies for controlling stem cell fate via morphogen sequestration.

Protein sequestration plays an essential role in maintaining stem cell populations in the native stem cell niche. Both pluripotent and adult stem cells require the sustained presentation of numerous bioactive growth factors and other soluble cues to potentiate cell fate decisions and morphogenic events. Consequently, methods of natural protein sequestration employed by the stem cell niche present attractive strategies for developing novel protein delivery vehicles and engineering biomimetic stem cell microenvironments that enhance morphogen bioactivity. In this review, we will explore the role of protein sequestration in the native stem cell niche and how it has inspired the design of several classes of materials that exploit natural protein sequestration to effectively maintain stem cell populations and direct stem cell fate. We will also highlight several recent developments in protein sequestering biomaterials, in which material strategies to sequester complex mixtures of endogenously secreted proteins are also being investigated.

Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions.

One of the significant challenges in bone tissue engineering is the integration of biomaterials designed to facilitate and stimulate mineralization with a simultaneously rapid rate of angiogenesis and vascularization of the tissue construct, a challenge complicated by our lack of knowledge of the interactions among key cell types and scaffold properties. This study compared functional activity of human bone marrow-derived stromal cells (hMSC) seeded onto a porous salt-leached poly(D,L-lactic acid) (PDLLA) scaffolds, with and without the incorporation of silk fibroin fibers and then further investigated their co-culture with human umbilical vein endothelial cells (HUVECs). Cell viability, proliferation, and alkaline phosphatase activity were measured for a range of time points in culture, with osteogenic and angiogenic marker immunolocalization and gene expression at selected stages. Our findings suggest that, despite similar porosity and pore size distribution exhibited by the PDLLA and PDLLA plus silk fibroin scaffolds, there were marked differences in cell distribution and function. In the absence of fibers, a highly osteogenic response was observed in hMSCs in the scaffolds co-cultured with endothelial cells, greater than that observed with hMSCs alone or in either of the scaffolds with fibers added. However, fiber presence clearly better supported endothelial cell cultures, as determined by greater levels of endothelial marker expression at both the gene and protein level after 3 weeks of culture. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. STATEMENT OF SIGNIFICANCE: A significant challenge in bone tissue engineering is to promote a rapid vascularization of the tissue construct in parallel to the extracellular matrix mineralization. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. Here we investigated cultures of hMSCs and HUVECs on a silk fibroin enhanced PDLLA scaffold, showing that the final output of this in vitro system is not the linear sum of the effects of the single variables. These results are of interest as they demonstrate how the addition of endothelial cells can affect hMSC phenotype and that the output can be further modulated by the introduction of silk fibroin fibers.

Contrast-enhanced microCT (EPIC-µCT) ex vivo applied to the mouse and human jaw joint.

OBJECTIVES: The temporomandibular joint (TMJ) is susceptive to the development of osteoarthritis (OA). More detailed knowledge of its development is essential to improve our insight into TMJ-OA. It is imperative to have a standardized reliable three-dimensional (3D) imaging method that allows for detailed assessment of both bone and cartilage in healthy and diseased joints. We aimed to determine the applicability of a contrast-enhanced microCT (µCT) technique for ex vivo research of mouse and human TMJs. METHODS: Equilibrium partitioning of an ionic contrast agent via µCT (EPIC-µCT) was previously applied for cartilage assessment in the knee joint. The method was ex vivo, applied to the mouse TMJ and adapted for the human TMJ. RESULTS: EPIC-µCT (30-min immersion time) was applied to mouse mandibular condyles, and 3D imaging revealed an average cartilage thickness of 110 ± 16 µm. These measurements via EPIC-µCT were similar to the histomorphometric measures (113 ± 19 µm). For human healthy OA-affected TMJ samples, the protocol was adjusted to an immersion time of 1 h. 3D imaging revealed a significant thicker cartilage layer in joints with early signs of OA compared with healthy joints (414.2 ± 122.6 and 239.7 ± 50.5 µm, respectively). A subsequent significant thinner layer was found in human joints with late signs of OA (197.4 ± 159.7 µm). CONCLUSIONS: The EPIC-µCT technique is effective for the ex vivo assessment of 3D cartilage morphology in the mouse as well as human TMJ and allows bone-cartilage interaction research in TMJ-OA.

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen-glycosaminoglycan scaffold in vivo.

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen-glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.

Localized 3D analysis of cartilage composition and morphology in small animal models of joint degeneration.

OBJECTIVE: Current histological scoring methods to evaluate efficacy of potential therapeutics for slowing or preventing joint degeneration are time-consuming and semi-quantitative in nature. Hence, there is a need to develop and standardize quantitative outcome measures to define sensitive metrics for studying potential therapeutics. The objectives of this study were to use equilibrium partitioning of an ionic contrast agent via Equilibrium Partitioning of an Ionic Contrast-Microcomputed tomography (EPIC-µCT) to quantitatively characterize morphological and compositional changes in the tibial articular cartilage in two distinct models of joint degeneration and define localized regions of interest to detect degenerative cartilage changes. MATERIALS AND METHODS: The monosodium iodoacetate (MIA) and medial meniscal transection (MMT) rat models were used in this study. Three weeks post-surgery, tibiae were analyzed using EPIC-µCT and histology. EPIC-µCT allowed measurement of 3D morphological changes in cartilage thickness, volume and composition. RESULTS: Extensive cartilage degeneration was observed throughout the joint in the MIA model after 3 weeks. In contrast, the MMT model showed more localized degeneration with regional thickening of the medial tibial plateau and a decrease in attenuation consistent with proteoglycan (PG) depletion. Focal lesions were also observed and 3D volume calculated as an additional outcome metric. CONCLUSIONS: EPIC-µCT was used to quantitatively assess joint degeneration in two distinct preclinical models. The MMT model showed similar features to human Osteoarthritis (OA), including localized lesion formation and PG loss, while the MIA model displayed extensive cartilage degeneration throughout the joint. EPIC-µCT imaging provides a rapid and quantitative screening tool for preclinical evaluation of OA therapeutics.

Prednisolone treatment and restricted physical activity further compromise bone of mdx mice.

OBJECTIVES: The purpose of this study was to determine the extent to which prednisolone treatment and restricted physical activity caused deleterious changes in inherently compromised mdx bone. METHODS: Four week-old male mdx mice (n=36) were treated for 8-wk either with or without prednisolone (0.8-1.3 mg/kg/d) and were housed in traditional or small cages (restricted activity). Tibial bone strength, geometry, and intrinsic material properties were assessed at the mid-shaft by three-point bending and micro-computed tomography (µCT). RESULTS: Three-point bending results showed that both prednisolone and restricted activity reduced bone strength (7%), however stiffness was only reduced in restricted-activity mice. µCT analyses showed that cortical bone area and cortical thickness were 13% smaller in restricted-activity mice, and may have accounted for their compromised bone strength. Intrinsic material properties, including volumetric bone mineral density (vBMD) and modulus of elasticity, were not impacted by either treatment, however, vBMD tended to be lower in restricted-activity mice (p=0.06). CONCLUSIONS: These data show that prednisolone treatment and restricted physical activity independently accentuate reductions in the strength and geometry of mdx bone, but do not influence intrinsic material properties.

Three years of alendronate treatment does not continue to decrease microstructural stresses and strains associated with trabecular microdamage initiation beyond those at 1 year.

UNLABELLED: The effects of a 3-year alendronate treatment on trabecular stresses/strains associated with microdamage initiation were investigated using finite element modeling (FEM). Severely damaged trabeculae in the low-dose treatment group were associated with increased stresses compared with the high-dose treatment group (p = 0.006) and approached significance in the control group (p = 0.02). INTRODUCTION: Alendronate, a commonly prescribed anti-remodeling agent, decreases fracture risk in the vertebrae, hip, and wrist of osteoporotic individuals. However, evaluation of microdamage accumulation in animal and human studies shows increased microdamage density relative to controls. Microstructural von Mises stresses associated with severe and linear damage have been found to decrease after 1 year of alendronate treatment. In the present study, stresses/strains associated with damage were assessed after 3 years of treatment to determine whether they continued to decrease with increased treatment duration. METHODS: Microdamaged trabeculae visualized with fluorescent microscopy were associated with stresses and strains obtained using image-based FEM. Stresses/strains associated with severe, diffuse, and linearly damaged and undamaged trabeculae were compared among groups treated for 3 years with an osteoporotic treatment dose of alendronate, a Paget's disease treatment dose of alendronate, or saline control. Architectural characteristics and mineralization were also analyzed from three-dimensional microcomputed tomography reconstructed images. RESULTS: Severely damaged trabeculae in the osteoporotic treatment dose group were associated with increased stress compared with the Paget's disease treatment dose group (p = 0.006) and approached significance compared to the control group (p = 0.02). Trabecular mineralization in severely damaged trabeculae of the low-dose treatment group was significantly greater compared to severely damaged trabeculae in the high-dose treatment and control group, suggesting that changes at the tissue level may play a role in these findings. CONCLUSIONS: Trabecular level stresses associated with microdamage do not continue to decrease with prolonged alendronate treatment. Changes in mineralization may account for these findings.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 µg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

Nondestructive assessment of sGAG content and distribution in normal and degraded rat articular cartilage via EPIC-microCT.

OBJECTIVE: The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-microCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models. DESIGN: After determination of an appropriate incubation time for the anionic contrast agent, EPIC-microCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 min of digestion with chondroitinase ABC. The EPIC-microCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora. RESULTS: Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 min with a 16.0% temporal increase (P<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4- to 8-weeks of age (P<0.01) and further increased by 2.5% from 8 to 16 weeks (P<0.05). After 60 min of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (P<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=-0.88, slope=-1.26, P<0.01, n=30) with EPIC-microCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation. CONCLUSIONS: EPIC-microCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

Quantitative assessment of articular cartilage morphology via EPIC-microCT.

OBJECTIVE: The objective of the present study was to validate the ability of Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-microCT) to nondestructively assess cartilage morphology in the rat model. DESIGN: An appropriate contrast agent (Hexabrix) concentration and incubation time for equilibration were determined for reproducible segmentation of femoral articular cartilage from contrast-enhanced microCT scans. Reproducibility was evaluated by triplicate scans of six femora, and the measured articular cartilage thickness was independently compared to thickness determined from needle probe testing and histology. The validated technique was then applied to quantify age-related differences in articular cartilage morphology between 4, 8, and 16-week-old (n=5 each) male Wistar rats. RESULTS: A 40% Hexabrix/60% phosphate buffered saline (PBS) solution with 30 min incubation was optimal for segmenting cartilage from the underlying bone tissue and other soft tissues in the rat model. High reproducibility was indicated by the low coefficient of variation (1.7-2.5%) in cartilage volume, thickness and surface area. EPIC-microCT evaluation of thickness showed a strong linear relationship and good agreement with both needle probing (r(2)=0.95, slope=0.81, P<0.01, mean difference 11+/-22 microm, n=43) and histology (r(2)=0.99, slope=0.97, P<0.01, mean difference 12+/-10 microm, n=30). Cartilage volume and thickness significantly decreased with age while surface area significantly increased. CONCLUSION: EPIC-microCT imaging has the ability to nondestructively evaluate three-dimensional articular cartilage morphology with high precision and accuracy in a small animal model.

Microscopic computed tomography imaging of the cerebral circulation in mice: feasibility and pitfalls.

This study evaluates the utility and practical limitations of microcomputerized X-ray tomography (CT) as a research tool for examination of the cerebral circulation in mice. Six micro CT angiograms of the circle of Willis (COW) from six mice were obtained by scanning whole head and brain specimen perfused with a radio-opaque silicone contrast agent. Two-dimensional volume rendered images were postprocessed from three-dimensional image datasets using a partially automated high-throughput model that generated 10 surface projections for each specimen. The image processing model employed a straightforward global thresholding and computerized component labeling software algorithm. Postprocessed images were analyzed and results correlated with microdissection. Micro CT demonstration of COW vessels and their branch anatomy was assessed. 71% of COW vessels were completely demonstrated, 26% were partially demonstrated, and 3% were not demonstrated. All cases of nondemonstration and most cases of partial demonstration resulted from scan coverage or postprocessing clip error. Thresholding effect caused pseudostenosis of 8% of COW vessels and accounted for a minority of partial demonstration cases. No imaging artifacts were caused by contrast extravasation or ineffective contrast perfusion. Volume averaging caused minor angioarchitectural distortion of 58% of COW vessels. Ninety-five percent of COW > or =50 microm and 52% of COW vessels <50 microm were correctly identified by micro CT. Micro CT of the murine COW using a high-throughput image processing model is feasible. Angioarchitectural distortion due to volume averaging and thresholding effect can occur and pathological findings should be confirmed.

Effects of treatment with parathyroid hormone 1-84 on quantity and biomechanical properties of thoracic vertebral trabecular bone in ovariectomized rhesus monkeys.

Osteoporosis is characterized by impaired bone quality leading to increased susceptibility to fracture, particularly of the thoracic spine. However, the lumbar spine is studied most commonly. We investigated the effects of 16 months of treatment with full-length parathyroid hormone (PTH) 1-84 (5, 10, or 25 microg/kg) on bone mineral density (BMD) and on architecture and biomechanical properties of trabecular bone at the thoracic spine of ovariectomized (OVX) adult rhesus monkeys and compared the results with those from the lumbar spine. At baseline, 9 months after surgery, dual-energy X-ray absorptiometric BMD at T9-T12 was 7% lower in OVX than in sham animals. All PTH(1-84) doses increased BMD to sham levels within 7 months. Micro-computed tomography of T10 vertebrae showed that trabecular bone volume and connectivity were higher in PTH(1-84)-treated animals than in sham controls, primarily through a significantly greater trabecular number. Peripheral quantitative computed tomography of trabecular bone cores from T11 and T12 confirmed that PTH(1-84) increased BMD. Compression testing of the cores showed that PTH(1-84) treatment increased stiffness, modulus, yield load, and yield stress to levels significantly greater than in sham animals, with the largest effect in the 10 microg/kg group (35-54% greater than in OVX controls). Thus, PTH(1-84) treatment increased BMD and the biomechanical properties of trabecular bone at the thoracic spine of OVX rhesus monkeys. The 10 microg/kg dose produced the greatest effect on trabecular strength, possibly because the highest dose stimulated bone remodeling excessively. Importantly, the changes observed were similar to those in lumbar vertebrae, thereby validating extrapolation of results from the lumbar to the thoracic spine.

Inducible regulation of Runx2-stimulated osteogenesis.

Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, this work describes a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant to developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.