Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia.

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.

Osteoinduction within adipose tissue fragments by heterodimeric bone morphogenetic Proteins-2/6 and -2/7 versus homodimeric bone morphogenetic protein-2: Therapeutic implications for bone regeneration.

BACKGROUND: Fragments of subcutaneous adipose tissue that have been genetically modified to express bone morphogenetic protein-2 (BMP-2) regenerate large segmental osseous lesions in rodents. Gene-activated adipose tissue can be implanted into osseous defects without prior cell extraction and cell culture. The present study aimed to explore whether the heterodimers BMP-2/6 or BMP-2/7 exceed the osteoinductive effect of BMP-2 on adipose tissue. METHODS: In an in vitro tissue culture system, freshly harvested rat subcutaneous adipose tissue was cultivated in the presence of either BMP-2 or BMP-2/6 or BMP-2/7 at a high (200 ng/ml) and low (50 ng/ml) concentration. Gene expression analysis as well as histological and immunohistochemical methods were applied to test for osteoinduction. RESULTS: A concentration of 200 ng/ml of homodimeric BMP-2 induced osteogenic differentiation most potently, showing more calcification and a higher expression level of bone markers than both concentrations of BMP-2/6 or -2/7. A concentration of 50 ng/ml of BMP-2 was a significantly stronger osteogenic inducer than both concentrations of BMP-2/6 and the low concentration of BMP-2/7. The most potent heterodimeric driver of osteoinduction was BMP-2/7 at a high concentration, demonstrating effects similar to those of BMP-2 at a low concentration. CONCLUSIONS: Homodimeric BMP-2 evoked osteoinduction within adipose tissue more potently and at a lower concentration than heterodimeric BMP-2/6 or BMP-2/7. This result agrees well with the fact that it might be easier to translate adipose grafts activated by homodimeric BMP-2 clinically. Preclinical in vivo gene transfer studies are necessary to confirm the results of the present study.

Gene activated adipose tissue fragments as advanced autologous biomaterials for bone regeneration: osteogenic differentiation within the tissue and implications for clinical translation.

Cost-effective, expedited approaches for bone regeneration are urgently needed in an ageing population. Bone Morphogenetic Proteins (BMPs) stimulate osteogenesis but their efficacy is impeded by their short half-life. Delivery by genetically modified cells can overcome this problem. However, cell isolation and propagation represent significant obstacles for the translation into the clinic. Instead, complete gene activated fragments of adipose tissue hold great potential for bone repair. Here, using an in-vitro culture system, we investigated whether adenoviral transduction with human BMP-2 can promote osteogenic differentiation within adipose tissue fragments. Osteoinduction in adipose tissue fragments was evaluated by quantitative reverse transcriptase polymerase chain reaction, immunohistology and histomorphometry. BMP-2 transduced adipose tissue synthesized BMP-2 protein over 30 days peaking by day six, which significantly promoted osteogenic differentiation as indicated by increased calcium depositions, up-regulation of bone marker genes, and bone-related protein expression. Our results demonstrate that cells within adipose tissue fragments can differentiate osteogenically after BMP-2 transduction of cells on the surface of the adipose tissue. BMP-2 gene activated adipose tissue represents an advanced osteo-regenerative biomaterial that can actively contribute to osteogenesis and potentially enable the development of a novel, cost-effective, one-step surgical approach to bone repair without the need for cell isolation.

Osteoinduction within BMP-2 transduced muscle tissue fragments with and without a fascia layer: implications for bone tissue engineering.

Bone can be engineered in vivo by implantation of gene-activated muscle tissue fragments. This expedited approach may be further improved by use of muscle tissue with attached fascia. The aim of this in vitro study was to provide an in depth comparison of the osteogenic differentiation capacity of muscle alone and muscle with fascia after BMP-2 transduction. Skeletal muscle tissue from rats was cut into pieces with and without a fascia layer on the surface. Adenoviral BMP-2 or GFP vectors were used for transduction. Osteogenic differentiation within the tissue fragments was evaluated and compared by qRT-PCR, alizarin red S staining, histomorphometry and immunohistology. Transduction efficiency and level of transgene expression were higher for muscle with fascia than muscle alone. Transduction with BMP-2 led to a significant upregulation of bone marker genes, proteins, and calcium deposition in both groups. Interestingly, histological evaluation revealed that osteoinduction did not occur within the fascia layer itself. The upregulation of bone marker genes in muscle with fascia was significantly lower after 2 weeks but similar after 4 weeks of in vitro culture in comparison to muscle alone. The fascia layer led to higher transduction efficiency and enhanced BMP-2 expression. Despite fascia's lower capacity for osteogenic differentiation, muscle implants may benefit from the fascia layer by the improved ability to deliver BMP-2. The presented data may contribute to the development of a novel, cost-effective, single-surgery bone engineering technology and encourage the evaluation of the osteoregenerative potential of muscle with fascia in an animal model.

Bone morphogenetic protein-2 is a stronger inducer of osteogenesis within muscle tissue than heterodimeric bone morphogenetic protein-2/6 and -2/7: Implications for expedited gene-enhanced bone repair.

BACKGROUND: Bone morphogenetic protein (BMP)-2 gene-activated muscle tissue fragments can regenerate large bone defects in preclinical animal models. The use of tissue fragments instead of isolated cells expedites gene-enhanced tissue engineering and may increase the possibility of clinical translation. The present in vitro study investigated whether the osteoinductive effect of BMP-2 on muscle tissue fragments can be enhanced using the heterodimers BMP-2/6 or BMP-2/7. METHODS: Skeletal muscle tissue fragments from rats were cultured in vitro for up to 20 days in normal medium, osteogenic medium or osteogenic medium supplemented with either a low (50 ng/ml) or high (200 ng/ml) concentration of recombinant human BMP-2, BMP-2/6 or BMP-2/7. Osteoinduction was evaluated by a quantitative reverse transcriptase-polymerase chain reaction, Alizarin red S staining, immunohistology and histomorphometry. RESULTS: Interestingly, BMP-2 was a significantly stronger inducer of osteogenic differentiation within muscle tissue than both heterodimers. Even the low concentration of BMP-2 elicited significantly higher levels of calcium deposition, bone-specific gene expression and protein production than the high concentration of both heterodimers. At the high concentration, BMP-2/7 had a significantly stronger osteogenic effect on muscle than BMP-2/6. CONCLUSIONS: The homodimer BMP-2 induced osteoblastogenesis in muscle faster, at a lower concentration and with a higher potency than the heterodimers BMP-2/6 or BMP-2/7. The findings of this in vitro study encourage bone repair by muscle implants in combination with BMP-2 single growth factor delivery, which might be beneficial with respect to clinical translation.

Recent advances in gene-enhanced bone tissue engineering.

The loss of bone tissue represents a critical clinical condition that is frequently faced by surgeons. Substantial progress has been made in the area of bone research, providing insight into the biology of bone under physiological and pathological conditions, as well as tools for the stimulation of bone regeneration. The present review discusses recent advances in the field of gene-enhanced bone tissue engineering. Gene transfer strategies have emerged as highly effective tissue engineering approaches for supporting the repair of the musculoskeletal system. By contrast to treatment with recombinant proteins, genetically engineered cells can release growth factors at the site of injury over extended periods of time. Of particular interest are the expedited technologies that can be applied during a single surgical procedure in a cost-effective manner, allowing translation from bench to bedside. Several promising methods based on the intra-operative genetic manipulation of autologous cells or tissue fragments have been developed in preclinical studies. Moreover, gene therapy for bone regeneration has entered the clinical stage with clinical trials for the repair of alveolar bone. Current trends in gene-enhanced bone engineering are also discussed with respect to the movement of the field towards expedited, translational approaches. It is possible that gene-enhanced bone tissue engineering will become a clinical reality within the next few years.

Gene-activated tissue grafts for sustained bone morphogenetic protein-2 delivery and bone engineering: Is muscle with fascia superior to muscle and fat?

Previously, we have presented an expedited strategy for sustained delivery of bone morphogenetic protein-2 (BMP-2) to bone lesions based on the implantation of gene-activated fat and muscle fragments. The aim of the present in vitro experiments was to evaluate the potential of muscle with fascia as a BMP-2 delivering osteo-regenerative implant in comparison to fat tissue and muscle alone. Subcutaneous fat, muscle, and muscle with fascia were harvested from Fischer 344 rats. The tissues were cut into small pieces and cultured for up to 90 days after direct transduction with adenoviral BMP-2 or green fluorescence protein vectors. Different vector doses were applied, and proliferation, long-term BMP-2 production, and osteogenic differentiation of the 3 different tissues were investigated in vitro. Muscle with fascia produced the largest amounts of BMP-2. Expression of the transgene was detected for up to 90 days. Proliferation was reduced with increased vector doses. Muscle with fascia showed a higher potential for osteogenic differentiation than fat, but it was not improved as compared to muscle alone. A dose of 4 × 108 plaque forming units of the adenoviral BMP-2 vector appeared to be the optimal dose for transduction of muscle with fascia. Because muscle with fascia produced higher amounts of BMP-2 as compared to muscle alone or fat tissue grafts, showing a high potential for osteogenic differentiation, it might represent an improved osteo-regenerative implant facilitating endogenous repair. Future studies should investigate the effect of muscle with fascia transduced with 4 × 108 plaque forming units on bone healing in vivo.

BMP-2 gene activated muscle tissue fragments for osteochondral defect regeneration in the rabbit knee.

BACKGROUND: Previously published data indicate that BMP-2 gene activated muscle tissue grafts can repair large bone defects in rats. This innovative abbreviated ex vivo gene therapy is appealing because it does not require elaborative and time-consuming extraction and expansion of cells. Hence, in the present study, we evaluated the potential of this expedited tissue engineering approach for regenerating osteochondral defects in rabbits. METHODS: Autologous muscle tissue grafts from female White New Zealand rabbits were directly transduced with an adenoviral BMP-2 vector or remained unmodified. Osteochondral defects in the medial condyle of rabbit knees were treated with either BMP-2 activated muscle tissue implants or unmodified muscle tissue or remained empty. After 13 weeks, repair of osteochondral defects was examined by biomechanical indentation testing and by histology/imunohistochemistry applying an extended O'Driscoll scoring system and histomorphometry. RESULTS: Biomechanical investigations revealed a trend towards slightly improved mechanical properties of the group receiving BMP-2 activated muscle tissue compared to unmodified muscle treatment and empty defect controls. However, a statistically significant difference was noted only between BMP-2 muscle and unmodified muscle treatment. Also, histological evaluation resulted in slightly higher histological scores and improved collagen I/II ratio without statistical significance in the BMP-2 treatment group. Histomorphometry indicated enhanced repair of subchondral bone after treatment with BMP-2 muscle, with a significantly larger bone area compared to untreated defects. CONCLUSIONS: Gene activated muscle tissue grafts showed potential for osteochondral defect repair. There is room for improvement via the use of appropriate growth factor combinations.

Osseous differentiation of human fat tissue grafts: From tissue engineering to tissue differentiation.

Conventional bone tissue engineering approaches require isolation and in vitro propagation of autologous cells, followed by seeding on a variety of scaffolds. Those protracted procedures impede the clinical applications. Here we report the transdifferentiation of human fat tissue fragments retrieved from subcutaneous fat into tissue with bone characteristics in vitro without prior cell isolation and propagation. 3D collagen-I cultures of human fat tissue were cultivated either in growth medium or in osteogenic medium (OM) with or without addition of Bone Morphogenetic Proteins (BMPs) BMP-2, BMP-7 or BMP-9. Ca2+ depositions were observed after two weeks of osteogenic induction which visibly increased when either type of BMP was added. mRNA levels of alkaline phosphatase (ALP) and osteocalcin (OCN) increased when cultured in OM alone but addition of BMP-2, BMP-7 or BMP-9 caused significantly higher expression levels of ALP and OCN. Immunofluorescent staining for OCN, osteopontin and sclerostin supported the observed real-time-PCR data. BMP-9 was the most effective osteogenic inducer in this system. Our findings reveal that tissue regeneration can be remarkably simplified by omitting prior cell isolation and propagation, therefore removing significant obstacles on the way to clinical applications of much needed regeneration treatments.

An expedited approach for sustained delivery of bone morphogenetic protein-7 to bone defects using gene activated fragments of subcutaneous fat.

BACKGROUND: Delivery of bone morphogenetic protein-7 (BMP-7) to bone defects can be improved by applying gene transfer methods. However, traditional ex vivo gene therapy approaches are cumbersome and costly, requiring the extraction and culturing of cells. Therefore, we evaluated a novel, expedited ex vivo BMP-7 gene transfer technology based on the use of fragments of subcutaneous fat tissue. METHODS: We created 5-mm mid-femoral bone defects in the right femora of 23 male, syngeneic Fischer 344 rats. Adipose tissue was harvested from the subcutaneous fat depot of two donor rats. Bone defects were treated with either unmodified fat (control group) or adenovirally BMP-7 transduced fat fragments (treatment group). Healing of bone defects was assessed by radiographs, microcomputed tomography (µCT) and histology at 6 weeks after the implantation of fat tissue fragments. RESULTS: Radiographs, µCT-imaging and histology revealed relevant bone formation in six out of 10 rats treated with BMP-7 activated fat grafts. Two of the defects were bridged. By contrast, femora of the control group receiving unmodified fat did not display signs of osseous healing. BMP-7 gene activated fat treatment led to a significantly higher bone volume (11.18 ± 9.48 mm(3) ) than treatment with unmodified fat grafts (3.19 ± 1.68 mm(3) ) (p = 0.008). CONCLUSIONS: Implantation of BMP-7 gene activated fat tissue fragments can elicit regeneration of large bone defects in rats and could become a clinically expeditious strategy for in vivo bone tissue engineering. However, gene expression must be improved in order to reliably induce osseous bridging of critical-size bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Gene-activated fat grafts for the repair of spinal cord injury: a pilot study.

BACKGROUND: Spinal cord injury (SCI) is a complex disease requiring a concerted multi-target approach. The most appropriate combination of therapeutic gene, cellular vehicle, and space filling scaffold still has to be determined. We present an approach that employs syngeneic adipose tissue serving as a three-dimensional biological implant, source of progenitor cells, and delivery system for therapeutic genes. In this pilot experiment, we evaluated the feasibility and short-term effects using gene-activated autologous fat grafts after SCI. METHODS: An experimental SCI model was established in syngeneic Fischer 344 rats by a T9-T10 hemimyelonectomy. Fat tissue was harvested from two donor rats. Animals were divided into four groups and treated with either (i) fat grafts activated by an adenoviral vector carrying the human NT-3 cDNA, (ii) or BDNF, (iii) or with untreated fat grafts or (iv) remained untreated. Animals were euthanized either 7 or 21 days after surgery, and spinal cord tissue was investigated by histological and immunohistochemical methods. RESULTS: NT-3 and BDNF were produced by gene-activated fat grafts for at least 21 days in vitro and in vivo. Fat tissue grafts remained stable at the site of implantation at 7 days and at 21 days. Neither BDNF-activated nor NT-3-activated fat graft had a detectable limiting effect on the neuronal degeneration. BDNF recruited microglia to perilesional site and attenuated their inflammatory response. CONCLUSIONS: Gene-activated syngeneic fat tissue serves as a three-dimensional biological material delivering therapeutic molecules to the site of SCI over an extended period of time. The BDNF-fat graft attenuated the inflammatory response. Whether these findings translate into functional recovery will require extended observation times.

Analysis of the autologous chondrocyte quality of matrix-based autologous chondrocyte implantation in the knee joint.

PURPOSE: Matrix based autologous chondrocyte implantation is an established method for treatment of full cartilage defects in the knee joint, but little is known about the influence of the implanted autologous chondrocyte quality and its clinical value. The aim of this study is to evaluate the influence of the gene expression of the implanted autologous chondrocytes on the clinical outcomes in the follow-up period of three years. METHODS: Biological parameters of the implanted chondrocytes were analysed histologically and immunohistologically. An analysis of the gene expression of the relevant chondrogenic dedifferentiation markers was performed as well. In order to detect the rate of apoptosis, we analysed specific apoptosis markers. To evaluate the clinical outcome, the International Knee Documentation Committee (IKDC) subjective score and the visual analogue scale (VAS) were used pre-operatively and after 0.5, one, two and three years. RESULTS: Significantly improved clinical scores were observed. The subjective IKDC score increased from 50.6 ± 20.7 points to 69.3 ± 26.1 points at three years (p = 0.001). The implanted chondrocytes showed a typical expression of the chondrogenic markers with a high rate of collagen type II expression without dedifferentiation. The analysed differentiation, dedifferentiation and apoptosis markers showed no influence on the clinical outcome. CONCLUSIONS: The implanted autologous chondrocytes have a high chondrogenic quality with a high rate of collagen type II expression without dedifferentiation. An influence of differentiation, de-differentiation and apoptosis marker gene expression on the clinical outcomes could not be found in a follow-up period of three years. LEVEL OF EVIDENCE: Case series; Level of evidence, 3.

The effect of BMP-7 gene activated muscle tissue implants on the repair of large segmental bone defects.

BACKGROUND: This study was conducted in order to investigate the effect of Bone Morphogenetic Protein-7 (BMP-7) transduced muscle cells on bone formation and to further develop an innovative abbreviated ex vivo gene therapy for bone repair. As conventional ex vivo gene therapy methods require an elaborative and time-consuming extraction and expansion of cells we evaluated an expedited approach. Fragments of muscle tissue were directly activated by BMP-7 cDNA and implanted into bone defects. METHODS: 25 male, syngeneic Fischer 344 rats were used in the present study. Muscle tissue was harvested from two donor rats and either transduced with an adenovirus carrying the BMP-7 cDNA or remained unmodified. 5mm osseous defects in the right femora of 23 rats were treated with either unmodified muscle tissue (control group) or BMP-7 activated muscle tissue (treatment group). Six weeks after surgery, rat femora were evaluated by radiographs, micro-computed tomography (µCT) and histology. RESULTS: Implantation of BMP-7 activated muscle grafts led to bony bridging in 5 out of 12 defects (41.7%) and to bone formation without bridging in 2 out of 12 defects. In 2 femoral defects of this group radiographs, µCT-imaging and histology did not reveal significant mineralization. Three animals of the BMP-7 treatment group had to be euthanized due to serious wound infection. The bone volume of the treatment group was significantly (p=0.007) higher compared to the control group. CONCLUSION: This study shows that BMP-7 gene activated muscle fragments have the potential to regenerate critical-size segmental bone defects in rats. However, further development of this promising expedited treatment modality is required to improve the healing rate and to investigate if the high infection rate is related to treatment with BMP-7 activated muscle grafts.

Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells.

PURPOSE: During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated. METHODS: A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-ß3 (TGF-ß3). The hMSC pellet cultures treated with TGF-ß3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR. RESULTS: SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio. CONCLUSIONS: SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Repair of large segmental bone defects: BMP-2 gene activated muscle grafts vs. autologous bone grafting.

BACKGROUND: Common cell based strategies for the treatment of osseous defects require the isolation and expansion of autologous cells. Since this makes such approaches time-consuming and expensive, we developed a novel expedited technology creating gene activated muscle grafts. We have previously shown that large segmental bone defects in rats can be regenerated by implantation of muscle tissue fragments activated by BMP-2 gene transfer. RESULTS: In the present study, we compared the bone healing capacities of such gene activated muscle grafts with bone isografts, mimicking autologous bone grafting, the clinical gold standard for treatment of bone defects in patients. Two of 14 male, syngeneic Fischer 344 rats used for this experiment served as donors for muscle and bone. Muscle tissue was harvested from both hind limbs and incubated with an adenoviral vector carrying the cDNA encoding BMP-2. Bone was harvested from the iliac crest and long bone epiphyses. Bone defects (5 mm) were created in the right femora of 12 rats and were filled with either BMP-2 activated muscle tissue or bone grafts. After eight weeks, femora were evaluated by radiographs, micro-computed tomography (µCT), and biomechanical testing. In the group receiving BMP-2 activated muscle grafts as well as in the bone-grafting group, 100% of the bone defects were healed, as documented by radiographs and µCT-imaging. Bone volume was similar in both groups and biomechanical stability of the two groups was statistically indistinguishable. CONCLUSIONS: This study demonstrates that treatment of large bone defects by implantation of BMP-2 gene activated muscle tissue leads to similar bone volume and stability as bone isografts, mimicking autologous bone grafting.

Improvement of tendon repair using muscle grafts transduced with TGF-ß1 cDNA.

Tendon rupture is a common injury. Inadequate endogenous repair often leaves patients symptomatic, with tendons susceptible to re-rupture. Administration of certain growth factors improves tendon healing in animal models, but their delivery remains a challenge. Here we evaluated the delivery of TGF-ß1 to tendon defects by the implantation of genetically modified muscle grafts. Rat muscle biopsies were transduced with recombinant adenovirus encoding TGF-ß1 and grafted onto surgically transected Achilles tendons in recipient animals. Tissue regenerates were compared to those of controls by biomechanical testing as well as histochemical and immunohistochemical analyses. Healing was greatly accelerated when genetically modified grafts were implanted into tendon defects, with the resulting repair tissue gaining nearly normal histological appearance as early as 2 weeks postoperatively. This was associated with decreased deposition of type III collagen in favour of large fibre bundles indicative of type I collagen. These differences in tendon composition coincided with accelerated restoration of mechanical strength. Tendon thickness increased in gene-treated animals at weeks 1 and 2, but by week 8 became significantly lower than that of controls suggesting accelerated remodelling. Thus localised TGF-ß1 delivery via adenovirus-modified muscle grafts improved tendon healing in this rat model and holds promise for clinical application.

Membrane-based cultures generate scaffold-free neocartilage in vitro: influence of growth factors.

Scaffold-free cultures provide promising potential in chondrogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a novel scaffold-free membrane-based culture system, in which hMSCs were cultivated on a cellulose acetate membrane filter at medium-gas interface, was evaluated for chondrogenesis under the addition of growth factors. Chondrogenic differentiation of hMSCs has been described in scaffold-free pellet cultures with good results. In our study membrane-based cultures (1 x 10(6) hMSCs) were produced, maintained at the medium-gas interface and cultured for 21 days. Results were compared with findings from standard pellet cultures (2.5 x 10(5) hMSCs). The effects of the following growth factors were examined: human transforming growth factor-beta(3) (TGF-beta(3)) +/- insulin-like growth factor-1 or +/- human fibroblast growth factor 2. After 3 weeks of culture, chondrogenesis was assessed by Safranin-O staining, immunohistochemistry, a dimethylmethylene blue dye binding assay for glycosaminoglycans, and quantitative real-time polymerase chain reaction for cartilage-specific proteins. Membrane-based cultures containing growth factors formed hemispherical structures with a large surface area (65 mm(2)). When removed from the membrane they showed a histologically smooth cartilage-like surface. Membrane-based cultures stained positive for Safranin-O and collagen type II and contained a high content of glycosaminoglycans. Expression of cartilage-specific markers like collagen type II, aggrecan, and SOX9 was observed under the addition of TGF-beta(3), whereas combinations of growth factors let to a significant increase of collagen type II expression. A markedly reduced expression of collagen type X was found in membrane-based cultures when only TGF-beta(3) was added. Pellet cultures showed similar results besides an increased expression of collagen type X and type II that were observed. Membrane-based cultures provide a differentiation system, comparable in chondrogenesis to pellet cultures, which is able to generate scaffold-free neocartilage. The key benefit factors of membrane-based cultures are a histologically smooth cartilage-like surface and reduced expression of collagen type X, both of which are suitable features for its future application in cartilage regeneration.

The repair of critical-sized bone defects using expedited, autologous BMP-2 gene-activated fat implants.

The repair of bone defects can be induced experimentally with bone morphogenetic protein-2 (BMP-2) producing fat-derived stem cells, but this ex vivo tissue engineering method requires the isolation and long-term culture of autologous cells. To develop an expedited bone repair strategy, we transferred BMP-2 cDNA directly to autologous fat tissue fragments that were held in culture for only 24 h before implantation. We evaluated the ability of such gene-activated fat grafts to regenerate large segmental bone defects in rats. Fat tissue was harvested from 2 of 35 male Fischer 344 rats used for this study. The fat tissue fragments were incubated with an adenoviral vector carrying the cDNA encoding either BMP-2 or green florescent protein (GFP), or they remained unmodified. According to their group, the segmental femoral bone defects of 33 rats were filled press fit with either BMP-2-activated fat tissue, GFP-transduced fat tissue, or unmodified fat tissue. Another control group remained untreated. Femora were evaluated by radiographs, microcomputed tomography, biomechanical torsional testing, and histology. Radiographically and histologically, 100% of the femora treated with BMP-2-activated fat grafts were bridged at 6 weeks after surgery. The femora of this group exceeded the bone volume and the biomechanical stability of intact, contralateral femora. Control defects receiving no treatment, unmodified fat tissue, or GFP-transduced fat were filled with fibrous or adipose tissue, as evaluated by histology. The use of BMP-2 gene-activated fat tissue grafts represents an expedited and effective bone repair strategy that does not require the extraction and expansion of stem cells.

Healing of large segmental bone defects induced by expedited bone morphogenetic protein-2 gene-activated, syngeneic muscle grafts.

Numerous preclinical studies have shown that osseous defects can be repaired by implanting bone morphogenetic protein (BMP)-2-transduced muscle cells. However, the drawback of this treatment modality is that it requires the isolation and long-term (approximately 3 weeks) culture of transduced autologous cells, which makes this approach cumbersome, time-consuming, and expensive. Therefore, we transferred BMP-2 cDNA directly to muscle tissue fragments that were held in culture for only 24 hr before implantation. We evaluated the ability of such gene-activated muscle grafts to induce bone repair. Two of 35 male, syngeneic Fischer 344 rats used in this study served as donors for muscle tissue. The muscle fragments remained unmodified or were incubated with an adenoviral vector carrying the cDNA encoding either green fluorescent protein (GFP) or BMP-2. Critical-size defects were created in the right femora of 33 rats and remained untreated or were filled (press fitted) with either unmodified muscle tissue or GFP-transduced muscle tissue or with BMP-2-activated muscle tissue. After 6 weeks, femora were evaluated by radiography, microcomputed tomography (muCT), histology, and biomechanical testing. Six weeks after implantation of BMP-2-activated muscle grafts, 100% of the bone defects were bridged, as documented by radiographs and muCT imaging, and showed formation of a neocortex, as evaluated by histology. Bone volumes of the femora repaired by BMP-2-transduced muscle were significantly (p = 0.006) higher compared with those of intact femora and the biomechanical stability was statistically indistinguishable. In contrast, control defects receiving no treatment, unmodified muscle, or GFP-transduced muscle did not heal. BMP-2 gene-activated muscle grafts are osteoregenerative composites that provide an expedited means of treating and subsequently healing large segmental bone defects.

Osteogenic potential of reamer irrigator aspirator (RIA) aspirate collected from patients undergoing hip arthroplasty.

Intramedullary nailing preceded by canal reaming is the current standard of treatment for long-bone fractures requiring stabilization. However, conventional reaming methods can elevate intramedullary temperature and pressure, potentially resulting in necrotic bone, systemic embolism, and pulmonary complications. To address this problem, a reamer irrigator aspirator (RIA) has been developed that combines irrigation and suction for reduced-pressure reaming with temperature modulation. Osseous particles aspirated by the RIA can be recovered by filtration for use as an autograft, but the flow-through is typically discarded. The purpose of this study was to assess whether this discarded filtrate has osteogenic properties that could be used to enhance the total repair potential of aspirate. RIA aspirate was collected from five patients (ages 71-78) undergoing hip hemiarthroplasty. Osseous particles were removed using an open-pore filter, and the resulting filtrate (230 +/- 200 mL) was processed by Ficoll-gradient centrifugation to isolate mononuclear cells (6.2 +/- 5.2 x 10(6) cells/mL). The aqueous supernatant contained FGF-2, IGF-I, and latent TGF-beta1, but BMP-2 was below the limit of detection. The cell fraction included culture plastic-adherent, fibroblastic cells that displayed a surface marker profile indicative of mesenchymal stem cells and that could be induced along the osteogenic, adipogenic, and chondrogenic lineages in vitro. When compared to outgrowth cells from the culture of osseous particles, filtrate cells were more sensitive to seeding density during osteogenic culture but had similar capacity for chondrogenesis. These results suggest using RIA aspirate to develop improved, clinically expeditious, cost-effective technologies for accelerating the healing of bone and other musculoskeletal tissues.