Bone morphogenetic protein 2-induced cellular chemotaxis drives tissue patterning during critical-sized bone defect healing: an in silico study

Critical-sized bone defects are critical healing conditions that, if left untreated, often lead to non-unions. To reduce the risk, critical-sized bone defects are often treated with recombinant human BMP-2. Although enhanced bone tissue formation is observed when BMP-2 is administered locally to the defect, spatial and temporal distribution of callus tissue often differs from that found during regular bone healing or in defects treated differently. How this altered tissue patterning due to BMP-2 treatment is linked to mechano-biological principles at the cellular scale remains largely unknown. In this study, the mechano-biological regulation of BMP-2-treated critical-sized bone defect healing was investigated using a multiphysics multiscale in silico approach. Finite element and agent-based modeling techniques were combined to simulate healing within a critical-sized bone defect (5 mm) in a rat femur. Computer model predictions were compared to in vivo microCT data outcome of bone tissue patterning at 2, 4, and 6 weeks postoperation. In vivo, BMP-2 treatment led to complete healing through periosteal bone bridging already after 2 weeks postoperation. Computer model simulations showed that the BMP-2 specific tissue patterning can be explained by the migration of mesenchymal stromal cells to regions with a specific concentration of BMP-2 (chemotaxis). This study shows how computational modeling can help us to further understand the mechanisms behind treatment effects on compromised healing conditions as well as to optimize future treatment strategies.

     

Size Does Matter: An Integrative In Vivo-In Silico Approach for the Treatment of Critical Size Bone Defects

Although bone has a unique restorative capacity, i.e., it has the potential to heal scarlessly, the conditions for spontaneous bone healing are not always present, leading to a delayed union or a non-union. In this work, we use an integrative in vivo - in silico approach to investigate the occurrence of non-unions, as well as to design possible treatment strategies thereof. The gap size of the domain geometry of a previously published mathematical model was enlarged in order to study the complex interplay of blood vessel formation, oxygen supply, growth factors and cell proliferation on the final healing outcome in large bone defects. The multiscale oxygen model was not only able to capture the essential aspects of in vivo non-unions, it also assisted in understanding the underlying mechanisms of action, i.e., the delayed vascularization of the central callus region resulted in harsh hypoxic conditions, cell death and finally disrupted bone healing. Inspired by the importance of a timely vascularization, as well as by the limited biological potential of the fracture hematoma, the influence of the host environment on the bone healing process in critical size defects was explored further. Moreover, dependent on the host environment, several treatment strategies were designed and tested for effectiveness. A qualitative correspondence between the predicted outcomes of certain treatment strategies and experimental observations was obtained, clearly illustrating the model''s potential. In conclusion, the results of this study demonstrate that due to the complex non-linear dynamics of blood vessel formation, oxygen supply, growth factor production and cell proliferation and the interactions thereof with the host environment, an integrative in silico-in vivo approach is a crucial tool to further unravel the occurrence and treatments of challenging critical sized bone defects.     

Porous Silk Scaffolds for Delivery of Growth Factors and Stem Cells to Enhance Bone Regeneration

Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.     

Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells

Background aimsMesenchymal stem/stromal cells (MSCs) are multipotent and self-renewing cells that are extensively used in tissue engineering. Adipose tissues are known to be the source of two types of MSCs; namely, adipose tissue–derived MSCs (ASCs) and dedifferentiated fat (DFAT) cells. Although ASCs are sometimes transplanted for clinical cytotherapy, the effects of DFAT cell transplantation on mandibular bone healing remain unclear.MethodsThe authors assessed whether DFAT cells have osteogenerative potential compared with ASCs in rats in vitro. In addition, to elucidate the ability of DFAT cells to regenerate the jaw bone, the authors examined the effects of DFAT cells on new bone formation in a mandibular defect model in (i) 30-week-old rats and (ii) ovariectomy-induced osteoporotic rats in vivo.ResultsOsteoblast differentiation with bone morphogenetic protein 2 (BMP-2) or osteogenesis-induced medium upregulated the osteogenesis-related molecules in DFAT cells compared with those in ASCs. BMP-2 activated the phosphorylation signaling pathways of ERK1/2 and Smad2 in DFAT cells, but minor Smad1/5/9 activation was noted in ASCs. The transplantation of DFAT cells into normal or ovariectomy-induced osteoporotic rats with mandibular defects promoted new bone formation compared with that seen with ASCs.ConclusionsDFAT cells promoted osteoblast differentiation and new bone formation through ERK1/2 and Smad2 signaling pathways in vitro. The transplantation of DFAT cells promoted new mandibular bone formation in vivo compared with that seen with ASCs. These results suggest that transplantation of ERK1/2-activated DFAT cells shorten the mandibular bone healing process in cytotherapy.     

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction

Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM).

Materials and Methods

The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5).

Results

PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.

Conclusion

Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.     

In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.     

Mechanistic,Mathematical Model to Predict the Dynamics of Tissue Genesis in Bone Defects via Mechanical Feedback and Mediation of Biochemical Factors

The link between mechanics and biology in the generation and the adaptation of bone has been well studied in context of skeletal development and fracture healing. Yet, the prediction of tissue genesis within - and the spatiotemporal healing of - postnatal defects, necessitates a quantitative evaluation of mechano-biological interactions using experimental and clinical parameters. To address this current gap in knowledge, this study aims to develop a mechanistic mathematical model of tissue genesis using bone morphogenetic protein (BMP) to represent of a class of factors that may coordinate bone healing. Specifically, we developed a mechanistic, mathematical model to predict the dynamics of tissue genesis by periosteal progenitor cells within a long bone defect surrounded by periosteum and stabilized via an intramedullary nail. The emergent material properties and mechanical environment associated with nascent tissue genesis influence the strain stimulus sensed by progenitor cells within the periosteum. Using a mechanical finite element model, periosteal surface strains are predicted as a function of emergent, nascent tissue properties. Strains are then input to a mechanistic mathematical model, where mechanical regulation of BMP-2 production mediates rates of cellular proliferation, differentiation and tissue production, to predict healing outcomes. A parametric approach enables the spatial and temporal prediction of endochondral tissue regeneration, assessed as areas of cartilage and mineralized bone, as functions of radial distance from the periosteum and time. Comparing model results to histological outcomes from two previous studies of periosteum-mediated bone regeneration in a common ovine model, it was shown that mechanistic models incorporating mechanical feedback successfully predict patterns (spatial) and trends (temporal) of bone tissue regeneration. The novel model framework presented here integrates a mechanistic feedback system based on the mechanosensitivity of periosteal progenitor cells, which allows for modeling and prediction of tissue regeneration on multiple length and time scales. Through combination of computational, physical and engineering science approaches, the model platform provides a means to test new hypotheses in silico and to elucidate conditions conducive to endogenous tissue genesis. Next generation models will serve to unravel intrinsic differences in bone genesis by endochondral and intramembranous mechanisms.     

Adjustable Stiffness,External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

Effect of Dual Treatment with SDF-1 and BMP-2 on Ectopic and Orthotopic Bone Formation

Purposes

The potent stem cell homing factor stromal cell-derived factor-1 (SDF-1) actively recruits mesenchymal stem cells from circulation and from local bone marrow. It is well established that bone morphogenetic protein-2 (BMP-2) induces ectopic and orthotopic bone formation. However, the exact synergistic effects of BMP-2 and SDF-1 in ectopic and orthotopic bone regeneration models have not been fully investigated. The purpose of this study was to evaluate the potential effects of simultaneous SDF-1 and BMP-2 treatment on bone formation.

Materials and Methods

Various doses of SDF-1 were loaded onto collagen sponges with or without BMP-2.These sponges were implanted into subcutaneous pockets and critical-size calvarial defects in C57BL/6 mice. The specimens were harvested 4 weeks post-surgery and the degree of bone formation in specimens was evaluated by histomorphometric and radiographic density analyses. Osteogenic potential and migration capacity of mesenchymal cells and capillary tube formation of endothelial cells following dual treatment with SDF-1 and BMP-2 were evaluated with in vitro assays.

Results

SDF-1-only-treated implants did not yield significant in vivo bone formation and SDF-1 treatment did not enhance BMP-2-induced ectopic and orthotopic bone regeneration. In vitro experiments showed that concomitant use of BMP-2 and SDF-1 had no additive effect on osteoblastic differentiation, cell migration or angiogenesis compared to BMP-2 or SDF-1 treatment alone.

Conclusions

These findings imply that sequence-controlled application of SDF-1 and BMP-2 must be further investigated for the enhancement of robust osteogenesis in bone defects.     

SDF-1 Enhances Wound Healing of Critical-Sized Calvarial Defects beyond Self-Repair Capacity

Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.     

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.     

Repair of rabbit ulna segmental bone defect using freshly isolated adipose-derived stromal vascular fraction

Background aimsStromal vascular fractions (SVF) from adipose tissue have heterogeneous cell populations, and include multipotent adipose-derived stem cells. The advantages of using of SVF include the avoidance of an additional culture period, a reduced risk of extensive cell contamination, and cost-effectiveness.MethodsUnilateral 20-mm mid-diaphyseal segmental defects in rabbit ulna were treated with one of the following: polylactic glycolic acid (PLGA) scaffold alone (group 1, control), a PLGA scaffold with undifferentiated SVF cells (group 2), or a PLGA scaffold with osteogenically differentiated SVF cells (group 3). At 8 weeks after implantation, five rabbits in each treatment group were killed to assess bone defect healing by plain radiography, quantitative microcomputed tomography and histology.ResultsThe SVF cells were well grown on PLGA scaffolds and expressed type I collagen and alkaline phosphatase (ALP). The intensity of ALP and OPN gene expressions in osteogenic medium culture were increased from 14 days to 28 days. In vivo evaluations at 8 weeks showed that treatment of SVF cells with or without osteogenic differentiation resulted in more bone formation in the critically sized segmental defects than PLGA scaffold alone. Osteogenically differentiated SVF cells significantly enhanced bone healing compared with undifferentiated SVF cells.ConclusionsAdipose-derived stromal SVF showed osteogenic potential in vitro. Accordingly, SVF could provide a cell source for bone tissue engineering. However, treatment with uncultured SVF cells on bone healing was not satisfactory in the in vivo animal model.     

Effects of Cell-Attachment and Extracellular Matrix on Bone Formation In Vivo in Collagen-Hydroxyapatite Scaffolds

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.     

Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.     

Predicting the external formation of a bone fracture callus: an optimisation approach

The formation of a fracture callus in vivo tends to form in a structurally efficient manner distributing tissues where mechanical stimulus persists. Therefore, it is proposed that the formation of a fracture callus can be modelled in silico by way of an optimisation algorithm. This was tested by generating a finite element model of a transversal bone fracture embedded in a large tissue domain which was subjected to axial, bending and torsional loads. It was found that the relative fragment motion induced a compressive strain field in the early callus tissue which could be utilised to simulate the formation of external callus structures through an iterative optimisation process of tissue maintenance and removal. The phenomenological results showed a high level of congruence with in vivo healing patterns found in the literature. Consequently, the proposed strategy shows potential as a means of predicting spatial bone healing phenomena for pre-clinical testing.     

牙周骨再生过程中BMP-1表达变化的临床意义

目的:观察在牙周骨再生过程中骨形态发生蛋白-1(BMP-1)的表达的变化情况及临床意义。方法:以我院收治的牙周骨缺损患者196例作为研究对象,采用常规的基础牙周治疗(龈上洁治彻底,龈下刮治,整平根面)处理后,通过引导组织再生术(GTR)技术对骨缺损进行修复的同时,植入Bio-oss人工骨材料和Bio-gide胶原膜。在治疗前和治疗后,分别测定患者血清BMP-1水平,分析BMP-1表达水平与牙周骨缺损修复的关系。结果:与治疗前相比,患者治疗后的牙周骨缺损均有所修复,所有患者在术后所有时间点的PPD和骨密度值与治疗前相比均显著改善(P0.05)。治疗6个月后,牙周骨密度出现小幅度下降,但显著高于治疗前,差异具有统计学意义(P0.05)。与治疗前相比,所有患者治疗后1 d、2 d、5 d、10 d、1个月、3个月、6个月血清BMP-1的水平均显著升高,差异具有统计学意义(P0.05),且治疗后BMP-1水平的变化与PPD呈显著负相关(P0.05),但与骨密度的相关性不显著(P0.05)。结论:血清BMP-1在牙周骨修复过程存在动态变化与患者牙周骨术后恢复相关。     

Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects

Background

Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect.

Methodology/Principal Findings

Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo.

Conclusions/Significance

Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Osteogenic comparison of expanded and uncultured adipose stromal cells

Background aimsAdipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC.MethodsAdipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard protocols or prepared by a commercial vendor using proprietary technology (proprietary stromal vascular fraction, SVFp). Cells were seeded on collagen sponges and maintained in osteogenic culture conditions for up to 21 days to assess osteogenic potential. The ability of each population to stimulate neovascularization and bone healing was determined upon implanting cell-loaded sponges into a rodent calvarial bone defect. Neovascularization was measured 3 weeks post-implantation, while bone formation was monitored over 12 weeks using in vivo microcomputed tomography (microCT).ResultsSVFp exhibited increased intracellular alkaline phosphatase activity compared with cultured ASC but proliferated minimally. Histologic analysis of explanted tissues demonstrated greater vascularization in defects treated with cultured ASC compared with SVFp. We detected increases in bone volume for defects treated with cultured cells while observing similar values for bone mineral density, regardless of cell type.ConclusionsThese results suggest that expanded ASC are advantageous for neovascularization and bone healing in this model compared with SVFp, and provide additional evidence of the utility of ASC in bone repair.