Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects.
ABSTRACT: Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.
Project description:<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
Project description:Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P<0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.
Project description:<h4>Background</h4>Periostin, an extracellular matrix protein, is expressed in bone, more specifically, the periosteum and periodontal ligaments, and plays a key role in formation and metabolism of bone tissues. Human adipose tissue-derived mesenchymal stem cells (hASCs) have been reported to differentiate into osteoblasts and stimulate bone repair. However, the role of periostin in hASC-mediated bone healing has not been clarified. In the current study, we examined the effect of periostin on bone healing capacity of hASCs in a critical size calvarial defect model.<h4>Methods and results</h4>Recombinant periostin protein stimulated migration, adhesion, and proliferation of hASCs in vitro. Implantation of either hASCs or periostin resulted in slight, but not significant, stimulation of bone healing, whereas co-implantation of hASCs together with periostin further potentiated bone healing. In addition, the number of Ki67-positive proliferating cells was significantly increased in calvarial defects by co-implantation of both hASCs and periostin. Consistently, proliferation of administered hASCs was stimulated by co-implantation with periostin in vivo. In addition, co-delivery of hASCs with periostin resulted in markedly increased numbers of CD31-positive endothelial cells and ?-SMA-positive arterioles in calvarial defects.<h4>Conclusions</h4>These results suggest that recombinant periostin potentiates hASC-mediated bone healing by stimulating proliferation of transplanted hASCs and angiogenesis in calvarial defects.
Project description:BACKGROUND:Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects. METHODS:Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction). RESULTS:Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression. CONCLUSIONS:Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.
Project description:Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P?<?0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P?<?0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P?<?0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze-thaw process.
Project description:An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.
Project description:The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2- and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor-based strategies in skeletal tissue engineering.
Project description:Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2, BMP-4, and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end, we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks, de novo bone formation was demonstrated by micro-CT imaging, histology, and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration, suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects, suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans, preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models.
Project description:There is an age-associated reduction in the bone healing activity of bone morphogenetic protein-2 (BMP-2) that is currently addressed by administering higher doses of BMP-2 in elderly patients. The unwanted medical complications from high dose BMP-2 motivated this investigation to determine whether the addition of a low dose of fibroblast growth factor 2 (FGF-2) could enhance the ability of a lower dose of BMP-2 to heal calvarial bone defects in old mice (18-20 months old). FGF-2 (5 ng) and BMP-2 (2 ?g) were administered by a controlled release two-phase biomaterial scaffold placed into the bone defect. FGF-2 released more rapidly and completely in vitro than BMP-2 (40% vs 2%). In vivo, both BMP-2 and FGF-2+BMP-2 groups formed more new bone in calvarial defects than scaffold alone (p < 0.001) or FGF-2 only groups (p < 0.01). The overall total volume of new bone was not statistically increased by the addition of FGF-2 to BMP-2 as measured by microCT, but the pattern of bone deposition was different. In old mice, but not young, there was enhanced bony fill in the central bone defect area when the BMP-2 was supplemented with FGF-2. Histological analysis of the center of the defect revealed an increased bone volume (%BV/TV (p = 0.004)) from the addition of FGF-2. These studies suggest that combining a low dose of FGF-2 with a low dose of BMP-2 has the potential to increase bone healing in old mice relative to BMP-2 alone.