Xiaoyu Da

Gene-Modified Adult Stem Cells Regenerate Vertebral Bone Defect in a Rat Model

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells

Bone regeneration achieved using mesenchymal stem cells (MSCs) and nonviral gene therapy holds great promise for patients with fractures seemingly unable to heal. Previously, MSCs overexpressing bone morphogenetic proteins (BMPs) were shown to differentiate into the osteogenic lineage and induce bone formation. In the present study, we evaluated the potential of osteogenic differentiation in porcine adipose tissue- and bone marrow-derived MSCs (ASCs and BMSCs, respectively) in vitro and in vivo when induced by nucleofection with rhBMP-2 or rhBMP-6. Our assessment of the in vivo efficiency of this procedure was made using quantitative micro-computed tomography (micro-CT). Nucleofection efficiency and cell viability were similar in both cell types; however, the micro-CT analyses demonstrated that in both ASCs and BMSCs, nucleofection with rhBMP-6 generated bone tissue faster and of higher volumes than nucleofection with rhBMP-2. RhBMP-6 induced more efficient osteogenic differentiation in vitro in BMSCs, and in fact, greater osteogenic potential was identified in BMSCs both in vitro and in vivo than in ASCs. On the basis of our findings, we conclude that BMSCs nucleofected with rhBMP-6 are superior at inducing bone formation in vivo than all other groups studied.

Human Induced Pluripotent Stem Cells Differentiate Into Functional Mesenchymal Stem Cells and Repair Bone Defects

Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self‐renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short‐term exposure of embryoid bodies to transforming growth factor‐β was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC‐derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow‐derived MSCs (BM‐MSCs). Ectopic injections of BMP6‐overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6‐overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self‐renewal without tumorigenic ability. Compared with BM‐MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture.

Human iPSCs Differentiate Into Functional MSCs and Repair Bone Defects

Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self‐renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short‐term exposure of embryoid bodies to transforming growth factor‐β was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC‐derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow‐derived MSCs (BM‐MSCs). Ectopic injections of BMP6‐overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6‐overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self‐renewal without tumorigenic ability. Compared with BM‐MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture.

PTH Promotes Allograft Integration in a Calvarial Bone Defect

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

Microbubble-enhanced, ultrasound-mediated BMP-6 gene delivery to endogenous progenitor cells induces rapid and efficient repair of critical-sized, nonunion bone fractures in mini-pigs. Bubbles and BMP-6 for bone repair Treatments for bone nonunions (fractures that fail to heal) include surgery and bone grafting. As an alternative to viral gene delivery, Bez et al. developed a two-step therapy. First, endogenous mesenchymal stem/progenitor cells were recruited to the bone nonunion by implanting a collagen sponge in the defect site. Two weeks later, bone morphogenetic protein-6 (BMP-6) plasmid DNA and microbubbles were injected into nonunions, and ultrasound was applied to oscillate the microbubbles, which helped the recruited progenitors take up the BMP-6. This therapy led to transient BMP-6 secretion, bone regeneration, and fracture healing over 6 weeks in critical-sized tibial nonunions in mini-pigs. More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro-computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis. The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7(th) day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7(th) and 10(th) postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Ultrasound-mediated transfection of endogenous stem cells for regenerative medicine

The oscillation of microbubbles has long been hypothesized to provide the opportunity to enhance gene delivery as a result of changes in membrane permeability; however, translationally-relevant therapeutic protocols have not yet been realized. We sought to develop and validate a protocol to transfect endogenous mesenchymal stem cells (MSCs) via the local injection of plasmids and microbubbles and the application of ultrasound. We apply this therapy in a pre-clinical model to solve an important clinical problem — that of healing segmental bone defects. More than two million bone-grafting procedures are performed each year using autografts or allografts and these standard of care therapies have substantial disadvantages.