Acute skeletal injury is necessary for human adipose-derived stromal cell-mediated calvarial regeneration.
ABSTRACT: 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:<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 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:Bone morphogenetic proteins (BMPs) have been widely used for bone repair in the craniofacial region. However, its high dose requirement in clinical applications revealed adverse effects and inefficient bone formation, along with high cost. Here, we report a novel osteoinductive strategy to effectively complement the osteogenic activity of BMP-2 using phenamil, a small molecule that can induce osteogenic differentiation via stimulation of BMP signaling. Treatment of adipose-derived stem cells (ASCs) with BMP-2 in combination with phenamil significantly promoted the in vitro osteogenic differentiation of ASCs. The efficacy of the combination strategy of phenamil+BMP-2 was further confirmed in a mouse calvarial defect model using scaffolds consisting of poly(lactic-co-glycolic acid) and apatite layer on their surfaces designed to slowly release phenamil and BMP-2. Six weeks after implantation, the scaffolds treated with phenamil+BMP-2 significantly promoted mouse calvarial regeneration as demonstrated by micro-computerized tomography and histology, compared with the groups treated with phenamil or BMP-2 alone. Moreover, the combination treatment reduced the BMP-2 dose without compromising calvarial healing efficacy. These results suggest promising complementary therapeutic strategies for bone repair in more efficient and cost-effective manners.
Project description:Adipose mesenchymal stem cells (ASCs) are considered as the promising seed cells for bone regeneration. However, the lower osteogenic differentiation capacity limits its therapeutic efficacy. Identification of the key molecules governing the differences between ASCs and BMSCs would shed light on manipulation of ASCs towards osteogenic phenotype. In this study, we screened semaphorin family members in ASCs and BMSCs and identified Sema3A as an osteogenic semaphorin that was significantly and predominantly expressed in BMSCs. The analyses in vitro showed that the overexpression of Sema3A in ASCs significantly enhanced the expression of bone-related genes and extracellular matrix calcium deposition, while decreasing the expression of adipose-related genes and thus lipid droplet formation, resembling a BMSCs phenotype. Furthermore, Sema3A modified ASCs were then engrafted into poly(lactic-co-glycolic acid) (PLGA) scaffolds to repair the critical-sized calvarial defects in rat model. As expected, Sema3A modified ASCs encapsulation significantly promoted new bone formation with higher bone volume fraction and bone mineral density. Additionally, Sema3A was found to simultaneously increase multiple Wnt related genes and thus activating Wnt pathway. Taken together, our study here identifies Sema3A as a critical gene for osteogenic phenotype and reveals that Sema3A-modified ASCs would serve as a promising candidate for bettering bone defect repair.
Project description:Although adipose-derived stem cells (ASCs) are an attractive cell source for bone tissue engineering, direct use of ASCs alone has had limited success in the treatment of large bone defects. Although bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors to promote osteogenic differentiation of ASCs, their clinical applications require supraphysiological dosage, leading to high medical burden and adverse side effects. In the present study, we demonstrated an alternative approach that can effectively complement the BMP activity to maximize the osteogenesis of ASCs without exogenous application of BMPs by regulating levels of antagonists and agonists to BMP signaling. Treatment of ASCs with the amiloride derivative phenamil, a positive regulator of BMP signaling, combined with gene manipulation to suppress the BMP antagonist noggin, significantly enhanced osteogenic differentiation of ASCs through increased BMP-Smad signaling in vitro. Furthermore, the combination approach of noggin suppression and phenamil stimulation enhanced the BMP signaling and bone repair in a mouse calvarial defect model by adding noggin knockdown ASCs to apatite-coated poly(lactic-coglycolic acid) scaffolds loaded with phenamil. These results suggest novel complementary osteoinductive strategies that could maximize activity of the BMP pathway in ASC bone repair while reducing potential adverse effects of current BMP-based therapeutics.Although stem cell-based tissue engineering strategy offers a promising alternative to repair damaged bone, direct use of stem cells alone is not adequate for challenging healing environments such as in large bone defects. This study demonstrates a novel strategy to maximize bone formation pathways in osteogenic differentiation of mesenchymal stem cells and functional bone formation by combining gene manipulation with a small molecule activator toward osteogenesis. The findings indicate promising stem cell-based therapy for treating bone defects that can effectively complement or replace current osteoinductive therapeutics.
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: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:The abnormal environment of type 2 diabetes mellitus (T2DM) leads to a substantial decrease in osteogenic function of stem cells. However, the gene sequence does not vary before and after disease for the patient. This phenomenon may be related to changes in osteogenesis-related gene expression caused by DNA methylation. In this study, we established T2DM models to extract adipose-derived stem cells (ASCs) for different gene identifications through DNA methylation sequencing. Specific fragments of methylation changes in the target gene (Calca) were identified by IGV analysis. CGRP was applied to compare the effects on ASCs-T2DM morphology via phalloidin staining, proliferation through CCK-8 assay, and osteogenic differentiation with osteogenic staining, qPCR, and repair of calvarial defect. Furthermore, 5-azacytidine (5-az) was used to intervene ASCs-T2DM to verify the relationship between the methylation level of the target fragment and expression of Calca. We found that the DNA methylation level of target fragment of Calca in ASCs-T2DM was higher than that in ASCs-C. CGRP intervention showed that it did not change the morphology of ASCs-T2DM but could improve proliferation within a certain range. Meanwhile, it could significantly enhance the formation of ALP and calcium nodules in ASCs-T2DM, increase the expression of osteogenesis-related genes in vitro, and promote the healing of calvarial defects of T2DM rat in a concentration-dependent manner. 5-az intervention indicated that the reduction of the methylation level in Calca target fragment of ASCs-T2DM indeed escalated the gene expression, which may be related to DNMT1. Taken together, the environment of T2DM could upregulate the methylation level in the promoter region of Calca and then decrease the Calca expression. The coding product of Calca revealed a promoting role for osteogenic differentiation of ASCs-T2DM. This result provides an implication for us to understand the mechanism of the decreased osteogenic ability of ASCs-T2DM and improve its osteogenic capacity.
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:Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFR? within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.