Evaluation of extruded starch foam for glucose-supplying biomaterials.

The survival rate of mesenchymal stem cells (MSC), a crucial factor in tissue engineering, is highly dependent on glucose supply. The purpose of this paper is to study the potential of starch foams as glucose suppliers. It is investigated through in vitro hydrolysis by amyloglucosidase in conditions that respect physiological constraints (37 °C and pH 7.4), including a duration of 21 days, and no stirring. Nine extruded starch foams with amylose contents ranging from 0 to 74 %, with various cell wall thicknesses (50 to 300 µm), and different crystallinities (0-30 %) were hydrolysed. These kinetics were fitted by a model which shows that the maximum rate of hydrolysis varies from 7 to 100 %, and which allows the rate of hydrolysis at 21 days to be calculated precisely. The results reveal the major role of amylose in glucose delivery kinetics, and the secondary roles of crystallinity and cell wall thickness of the foams. Additional hydrolysis of starch films revealed that thickness positively influences the amylose chain reorganisation during hydrolysis, which, in slows down and limits glucose delivery. A simple glucose delivery kinetics analysis procedure is proposed to select samples for testing as MSC glucose suppliers.

Human mesenchymal stem cell responses to hydrostatic pressure and shear stress.

The effects of mechanical stimuli to which cells are exposed in vivo are, at best, incompletely understood; in this respect, gene-level information regarding cell functions which are pertinent to new tissue formation is of special interest and importance in applications such as tissue engineering and tissue regeneration. Motivated by this need, the present study investigated the early responses of human mesenchymal stem cells (hMSCs) to intermittent shear stress (ISS) and to cyclic hydrostatic pressure (CHP) simulating some aspects of the biological milieu in which these cells exist in vivo. Production of nitric oxide (NO) and mRNA expression of several known mechanosensitive genes as well as ERK1/2 activation in the hMSC response to the two mechanical stimuli tested were monitored and compared. NO production depended on the type of the mechanical stimulus to which the hMSCs were exposed and was significantly higher after exposure to ISS than to CHP. At the conditions of NO peak release (i.e., at 0.7 Pa for ISS and 50,000 Pa for CHP), ISS was more effective than CHP in up-regulating mechanosensitive genes. ERK1/2 was activated by ISS but not by CHP. The present study is the first to report that PGTS2, IER3, EGR1, IGF1, IGFBP1, ITGB1, VEGFA and FGF2 are involved in the response of hMSCs to ISS. These findings establish that, of the two mechanical stimuli tested, ISS is more effective than CHP in triggering expression of genes from hMSCs which are bioactive and pertinent to several cell functions (such as cell differentiation and release of specific growth factors and cytokines) and also to tissue-related processes such as wound healing.

Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction.

BACKGROUND: Tissue engineering strategies include both cell-based and cell homing therapies. Ligamentous tissues are highly specialized and constitute vital components of the musculoskeletal system. Their damage causes significant morbidity and loss in function. HYPOTHESIS: The aim of this study is to analyze tendinous graft integration, cell repopulation and ligamentization by using GFP+/- allografts in GFP+/- transgenic New Zealand white (NZW) rabbits. MATERIAL AND METHODS: Graft implantation was designed to closely mimic anterior cruciate ligament (ACL) repair surgery. Allografts were implanted in 8 NZW rabbits and assessed at 5 days, 3 weeks and 6 weeks through: (1) arthroCT imaging, (2) morphological analysis of the transplanted allograft, (3) histological analysis, (4) collagen type I immunochemistry, and (5) GFP cell tracking. Collagen remodeling was appreciated at 3 and 6 weeks. RESULTS: Graft repopulation with host cells, chondrocyte-like cells at the tendon-bone interface and graft corticalization in the bone tunnels were noticed at 3 weeks. By contrast we noticed a central necrosis aspect in the allografts intra-articularly at 6 weeks with a cell migration towards the graft edge near the synovium. DISCUSSION: Our study has served to gain a better understanding of tendinous allograft bone integration, ligamentization and allograft repopulation. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. Future studies may elucidate whether cell repopulation occurs with pre-differentiated or progenitor cells. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. LEVEL OF EVIDENCE: Level V (animal study).

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer.

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

Strategies for improving the efficacy of bioengineered bone constructs: a perspective.

Bioengineered bone scaffolds are intended for use in large bone defects. Successful bone constructs should stimulate and support both the onset and the continuance of bone ingrowth. In an attempt to improve their performance and to compete with the one of autologous bone grafts, a growing symbiosis at the biological and material level is required. Recent advances have been made to further exploit the osteogenic potential of MSCs in scaffold development. Current research encompasses new strategies for reducing cell death after implantation and the manufacturing of tailored, instructive scaffolds.

Survival and function of mesenchymal stem cells (MSCs) depend on glucose to overcome exposure to long-term, severe and continuous hypoxia.

Use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for various diseases but has generated marginally successful results. A consistent finding of most studies is massive death of transplanted cells. The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long-term (12 days), severe (pO(2) < 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell-driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO(2) , hypoxia inducible factor-1α expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO(2) ) to ischemic (low pO(2) and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.

[Use of the induced membrane technique for the treatment of bone defects in the hand or wrist, in emergency]. / Pertes de substance osseuse à la main et au poignet traitées en urgence par technique de la membrane induite (technique de Masquelet).

A prospective study is reported concerning 11 cases of bone defect of the hand and wrist treated by the induced membrane technique. Ten men and one woman with an average age of 49 yrs (17-72) sustained a high-energy trauma with severe mutilation of digit and hand but with intact pulp. Eight cases of open finger fractures with composite loss of substance and three cases of bone and joint infection (thumb, wrist, fifth finger) were included. All cases were treated by the induced membrane technique which consists in stable fixation, flap if necessary, and in filling the bone defect by a cement methyl methacrylate polymere (PMMA) spacer. A secondary procedure at two months is needed where the cement is removed and the void is filled by cancellous bone. The key point of this induced membrane technique is to respect the foreign body membrane which formed around the cement spacer creating a biologic chamber. Bone union was evaluated prospectively by X-ray and CT scan by a surgeon not involved in the treatment. Failure was defined as non-union at one year, or uncontrolled sepsis at one month. Two cases failed to achieve bone union. No septic complications occurred and all septic cases were controlled. In nine cases, bone union was achieved within four months (three to 12). Evidence of osteoid formation was determined by a bone biopsy in one case. Masquelet first reported 35 cases of large tibial non-union defects treated by the induced membrane technique. The cement spacer promotes foreign body membrane induction constituting a biological chamber. Works on animal models reported by Pellissier and Viateau demonstrated membrane properties: secretion of growths factors (VEGF, TGF beta1, BMP2) and osteoinductive cellular activity. The induced membrane seems to mimic a neoperiosteum. This technique is useful in emergency or septic conditions where bone defects cannot be treated by shortening. It avoids microsurgery and is limited by availability of cancellous bone.

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection.

Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm(-1) with pre-incubation in Spinner's minimum essential medium buffer at 22 degrees C. Under these conditions beta-galactosidase expression was achieved in 29+/-3% of adherent cells 48 h post transfection. The kinetics of beta-galactosidase activity revealed maintenance of beta-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

Engineering of implantable cartilaginous structures from bone marrow-derived mesenchymal stem cells.

Fabrication of implantable cartilaginous structures that could be secured in the joint defect could provide an alternative therapeutic approach to prosthetic joint replacement. Herein we explored the possibility of using biodegradable hydrogels in combination with a polyglycolic acid (PGA) scaffold to provide an environment propitious to mesenchymal stem cells (MSCs) chondrogenic differentiation. We examined the influence of type I collagen gel and alginate combined with PGA meshes on the extracellular matrix composition of tissue-engineered transplants. MSCs were isolated from young rabbits, expanded in monolayers, suspended in each hydrogel, and loaded on PGA scaffolds. All constructs (n=48) were cultured in serum-free medium containing transforming growth factor beta-1, under dynamic conditions in specially designed bioreactors for 3-6 weeks. All cell-polymer constructs had a white, shiny aspect, and retained their initial size and shape over the culture period. Their thickness increased substantially over time, and no shrinkage was observed. All specimens developed a hyalin-like extracellular matrix containing glycosaminoglycans (GAGs) and type II collagen, but significant differences were observed among the three different groups. In PGA/MSCs and collagen-PGA/MSCs constructs, the cell growth phase and the chondrogenic differentiation phase of MSCs occurred during the first 3 weeks. In alginate-PGA/MSCs constructs, cells remained round in the hydrogel and cartilage extracellular matrix deposition was delayed. However, at 6 weeks, alginate-PGA/MSCs constructs exhibited higher contents of GAGs and lower contents of type I collagen. These results suggest that the implied time for the transplantation of in vitro engineered constructs depends, among other factors, on the nature of the scaffold envisioned. In this study, we demonstrated that the use of a composite hydrogel-PGA scaffold supported the in vitro growth of implantable cartilaginous structures cultured in a bioreactor system.

Embedding of bone samples in methylmethacrylate: a suitable method for tracking LacZ mesenchymal stem cells in skeletal tissues.

Considerable research has been focused on the use of bone marrow-derived mesenchymal stem cells (MSCs) for the repair of non-unions and bone defects. To date, the question of whether transplanted MSCs survive and engraft within newly formed tissue remains unresolved. The development of an easy and reliable method that would allow cell fate monitoring in transplant recipients is a pressing concern for the field of tissue engineering. To demonstrate the presence of transplanted cells in newly formed bone, we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in vivo. MSCs were isolated from transgenic lacZ mice, seeded onto bioabsorbable collagen sponges, and transplanted to repair a calvarial defect in nude rats. As a preliminary step, the histological procedure was adapted to optimize the detection of LacZ cells in bone tissue embedded in methylmethacrylate (MMA). Four fixatives and four fixation times were evaluated. Among all the fixatives tested, 2% formaldehyde/0.2% glutaraldehyde at 4C for 4 days gave the best results for X-gal staining at pH 7.4 on both cell cultures and bone explants. All fixatives were effective for immunodetection of beta-gal. In the chimeric LacZ/nude rat animal model, MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair and the neovascularization of the bone defect. LacZ is a suitable phenotypic marker to track MSCs in skeletal tissues embedded in MMA.

De novo reconstruction of functional bone by tissue engineering in the metatarsal sheep model.

Large bone defects are still a challenge to orthopedic surgeons. In this study, a massive bone defect with a clinically relevant volume was efficiently reconstructed by transplanting an engineered bone in which mesenchymal stem cells (MSCs) expanded in autologous serum (AS) were combined with a porous scaffold. In the first step, we established that the way in which the MSCs are distributed over the scaffold affects the ultimate bone-forming ability of the transplant: constructs consisting of a natural coral scaffold and a pseudo-periosteal layer of MSCs surrounding the implant (coral-MSC3D) formed significantly more bone than constructs in which the MSCs were distributed throughout the implant (p = 0.01). However, bone healing occurred in only one sheep, owing to the high resorption rate of natural coral scaffold. To overcome this problem, constructs in which MSCs were combined with a porous coralline-based hydroxyapatite (CHA) scaffold having the same architecture as natural coral but a lower resorption rate were prepared. After their implantation, these constructs were found to have the same osteogenic potential as autologous bone grafts in terms of the amount of newly formed bone present at 4 months (p = 0.89) and to have been completely replaced by newly formed, structurally competent bone within 14 months. Nevertheless, although the rate of bone healing was strikingly improved when CHA-MSC3D constructs were used (five of seven animals healed) as compared with the coral-MSC3D construct (one of seven healed), it was still less satisfactory than that obtained with autografts (five of five healed).

Engineering bone: challenges and obstacles.

Repair of large bone defects is still a challenge for the orthopaedic, reconstructive and maxillo-facial surgeon. Availability of pluripotent stem cells from either autologous or allogenic sources and the potential of inducing the osteogenic phenotype is motivating exploration and development of custom-tailored materials known as "bioengineered bone constructs". In such cases, the clinical scenario involves either expansion of stem cells in monolayer and loading them into a porous scaffold prior to surgery or direct cell expansion within the scaffold, and implanting this novel construct back into the donor patient. In this review, we delineate, from an engineering perspective, the progress that has been made to date and the challenges remaining in successfully translating this promising (but not yet definitively established) approach from bench to the bed site.

[Therapeutic application of mesenchymal stem cells in orthopaedics]. / Utilisation thérapeutique des cellules souches en orthopédie.

Stem cell therapy of skeletal tissues involves the transplantation of stem cells to the tissues that have been damaged by injury or disease. Although these cells can be derived from embryos, the preferred source of skeletal stem cells is the bone marrow as it contains adult stem cells that can be easily driven towards a bone phenotype. More recently, cells with similar potentialities have also been derived from adipose tissue, muscle, or blood. A biomaterial (ceramics or polymers) is often required as a scaffold to promote cell adhesion, proliferation and differentiation as well as encourage vascular invasion and ultimately new bone formation. The first clinical studies are encouraging and suggests that stem cell therapy could be a prime method for bone reconstruction.

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye.

Recent advances in the field of tissue engineering have culminated in new tissue substitutes that combine a biomaterial and precursor cells. The effectiveness of these materials is generally assessed in animals, but few studies explore the fate of the transplanted cells in vivo, despite its paramount importance for understanding the function of the engineered tissues. Current methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a new method for irreversible labeling of cells to track their fate in vivo. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 microM CM-Dil in 30 min. The CM-Dil was not cytotoxic and did not affect cell proliferation in vitro. Cells could be monitored for up to 30 days when placed in a coral scaffold and implanted intramuscularly or in a bony site. However, the fluorescence intensity decreased roughly in parallel with the number of cell divisions. This fact needs to be taken into account during the design and interpretation of experiments. We believe that this technique is also of interest for other cell types.

Tissue-engineered bone regeneration.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

[Biomaterials and osseous regeneration]. / Biomatériaux et régénération osseuse.

The autologous bone graft is commonly used for the repair of bony defects, but its resorption is unpredictable, and there is an inherent morbidity of the donor site. There is a wide range of biomaterials that could be used as bone substitutes, depending on their bioactivity. Among bioactive materials, bioglasses present a linkage between their reactive surface and the adjacent bone although they cannot be colonized by bony ingrowth, moreover their fragility and resorption as particles limit their use. The osteoconductive biomaterials are either represented by the synthetized ceramics, such as hydroxyapatite (HA) or tricalcium phosphate (TCP), or either natural coral and the derived biomaterials of bony matrix. Coral exoskeleton or TCP are highly resorbable, but pure HA is only slightly. Bony ingrowth in osteoconductive materials is limited to the periphery of the implant which does not make it suitable for the repair of large defects. Research is focused on the adjunction of a biologically active substance to the osteoconductive matrix in order to enhance bony ingrowth. Osteoinductive materials such as bone growth factors in combination with a carrier can promote bone healing, especially when bone morphogenetic protein (BMP) is used. Nevertheless, even if their efficacy is demonstrated, their inocuity has not been totally confirmed. Furthermore, the dose used are far superior than in the physiological pathways. Hybrid biomaterials combine an osteoconductive carrier with bone marrow cells. Bone cell cultures could amplify to almost any extent the number of osteogenic cells for such a biomaterial. Bone substitutes will certainly be used in the future to repair bony defects.

Osteogenesis with coral is increased by BMP and BMC in a rat cranioplasty.

Autologous bone marrow cells (BMC), bone morphogenetic protein (BMP) and natural coral exoskeleton (CC) were used to enhance the repair of large skull bone defects in a craniotomy model. Nine millimeter calvarial defects were created in adult rats and were either left empty (control defects) or implanted with CC alone, CC-BMC, CC-BMP, or CC-BMC-BMP. After 1 or 2 months, osteogenesis was insufficient to allow union when defects were left empty or filled with CC. Addition of BMC alone to CC had no positive influence on osteogenesis at any time and increased CC resorption at 2 months (0.1 +/- 0.1 mm2 versus 0.5 +/- 0.3 mm2). In contrast addition of BM P or BM P/BMC to CC led to a significant increase in osteogenesis and allowed bone union after 1 month. At 2 months, the combination of CC-BM P-BMC was the most potent activator of osteogenesis. Filling a defect with CC-BMP-BMC resulted in significantly increased bone surface area (11 +/- 2.7 mm2) in comparison to filling a defect with CC-BMP (7.0 +/- 1.4 mm2), CC-BMC (3.5 +/- 1.1 mm2) or CC (4.5 +/- 0.4 mm2). CC resorption was significantly decreased in the presence of BMP with or without BMC at both times. These data are in accordance with the presence of progenitor cells in bone marrow that are inducible by BMP to the osteogenic pathway in a cranial site. The increase in material resorption in defects filled with CC-BMC could suggest that cells from the granulocyte-macrophage lineage survived the grafting procedure and were still active after 2 months.

Coral grafting supplemented with bone marrow.

Limited success in regenerating large bone defects has been achieved by bridging them with osteoconductive materials. These substitutes lack the osteogenic and osteoinductive properties of bone autograft. A direct approach would be to stimulate osteogenesis in these biomaterials by the addition of fresh bone-marrow cells (BMC). We therefore created osteoperiosteal gaps 2 cm wide in the ulna of adult rabbits and either bridged them with coral alone (CC), coral supplemented with BMC, or left them empty. Coral was chosen as a scaffold because of its good biocompatibility and resorbability. In osteoperiosteal gaps bridged with coral only, the coral was invaded chiefly by fibrous tissue. It was insufficient to produce union after two months. In defects filled with coral and BMC an increase in osteogenesis was observed and the bone surface area was significantly higher compared with defects filled with coral alone. Bony union occurred in six out of six defects filled with coral and BMC after two months. An increase in the resorption of coral was also observed, suggesting that resorbing cells or their progenitors were present in bone marrow and survived the grafting procedure. Our findings have shown that supplementation of coral with BMC increased both the resorption of material and osteogenesis in defects of a clinical significance.