A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model.

OBJECTIVES: To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects. MATERIALS AND METHODS: Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal-Wallis tests. Data were expressed as mean and standard deviation. RESULTS: A two-fold increaseof newly formed bone volume was observed for Acropora-TECs when compared with Porites-TECs (14 sd 1089 mm3versus 782 sd 507 mm3; p = 0.09). Bone union was consistent with autograft (1960 sd 518 mm3). The kinetics of bioresorption and bioresorption rates at four months were different for Acropora-TECs and Porites-TECs (81% sd 5% versus 94% sd 6%; p = 0.04). In comparing the defects that healed with those that did not, we observed that, when major bioresorption of coral at two months occurs and a scaffold material bioresorption rate superior to 90% at four months is achieved, bone nonunion consistently occurred using coral-based TECs. DISCUSSION: Bone regeneration in critical-size defects could be obtained with full bioresorption of the scaffold using coral-based TECs in a large animal model. The superior performance of Acropora-TECs brings us closer to a clinical application, probably because of more suitable bioresorption kinetics. However, nonunion still occurred in nearly half of the bone defects.Cite this article: A. Decambron, M. Manassero, M. Bensidhoum, B. Lecuelle, D. Logeart-Avramoglou, H. Petite, V. Viateau. A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model. Bone Joint Res 2017;6:208-215. DOI: 10.1302/2046-3758.64.BJR-2016-0236.R1.

Assessment of a polyelectrolyte multilayer film coating loaded with BMP-2 on titanium and PEEK implants in the rabbit femoral condyle.

UNLABELLED: The aim of this study was to evaluate the osseointegration of titanium implants (Ti-6Al-4V, noted here TA6V) and poly(etheretherketone) PEEK implants induced by a BMP-2-delivering surface coating made of polyelectrolyte multilayer films. The in vitro bioactivity of the polyelectrolyte film-coated implants was assessed using the alkaline phosphatase assay. BMP-2-coated TA6V and PEEK implants with a total dose of 9.3µg of BMP-2 were inserted into the femoral condyles of New Zealand white rabbits and compared to uncoated implants. Rabbits were sacrificed 4 and 8weeks after implantation. Histomorphometric analyses on TA6V and PEEK implants and microcomputed tomography on PEEK implants revealed that the bone-to-implant contact and bone area around the implants were significantly lower for the BMP-2-coated implants than for the bare implants. This was confirmed by scanning electron microscopy imaging. This difference was more pronounced at 4weeks in comparison to the 8-week time point. However, bone growth inside the hexagonal upper hollow cavity of the screws was higher in the case of the BMP-2 coated implants. Overall, this study shows that a high dose of BMP-2 leads to localized and temporary bone impairment, and that the dose of BMP-2 delivered at the surface of an implant needs to be carefully optimized. STATEMENT OF SIGNIFICANCE: The presentation of growth factors from material surfaces currently presents significant challenges in academia, clinics and industry. Applying osteoinductive factors to different types of implants, made of metals or polymers, may improve bone repair in difficult situations. Here, we show the effects of an osteoinductive coating made of polyelectrolyte multilayer films on two widely used materials, titanium TA6V alloys and PEEK implants, which were implanted in the rabbit femoral condyle. We show that a too high dose of BMP-2 delivered from the screw surface has a negative short-term effect on bone regeneration in close vicinity of the screw surface. In contrast, bone formation was increased at early times in the empty spaces around the screw. These results highlight the need for future dose-dependence studies on bone formation in response to osteoinductive coatings.

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.

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.

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.

An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct.

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP-2 (0.5-100ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24h vs. 3-6 days, respectively, of BMP treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGFbeta1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells.

[Use of growth factors in the repair of bone]. / Utilisation de facteurs de croissance pour la réparation osseuse.

Osteoformation is induced by numerous growth factors that play an important role in bone repair such as fracture healing. They may serve as therapeutic agent in the treatment of squeletal injuries in the orthopeadic and maxillo-facial fields. Among these proteins, Bone Morphogenetic Proteins (BMP) are the only known osteoinductive growth factors. Unfortunately, they are highly susceptible to proteolysis in vivo and require a suitable delivery system to potentiate their biological activity in a local, controlled and durable manner. In this aim, three options are under investigations: (i) osteoinductive materials made of appropriate carrier to release the protein in situ, (ii) in vivo gene therapy in which the gene is directly transfected in cells of the patient or (iii) ex vivo gene therapy in which cells are harvested from the patient, transfected with DNA in culture and then implanted in the defect. These different kinds of BMP delivery will be discussed.

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.

Carboxymethyl benzylamide sulfonate dextrans (CMDBS), a family of biospecific polymers endowed with numerous biological properties: a review.

The functionalized dextrans termed carboxymethyl benzylamide sulfonate dextran (CMDBS) represent a family encompassing a wide range of polymers. These soluble macromolecular compounds, which are substituted with specific chemical functional groups, are designed to interact with living systems. By analogy with glycosaminoglycan heparin, a natural highly charged anionic polysaccharide that exerts a variety of biological effects, we postulated that CMDBS compounds also possess binding sites capable of specific interactions with biological constituents, depending on the overall composition of the polymer. The synthesis and heparin-like properties of these CMDBS have been extensively investigated. Thus, it appears that dextran derivatives can mimic the action of heparin in regard to its interactions with antithrombin and serine proteases involved in blood coagulation. Other derivatives interact with various components of the immune system or with adhesive proteins such as fibronectin in modulating the proliferation of Staphylococcus aureus. Because they are able to stimulate wound healing in various in vivo models, these polysaccharides may also constitute a family of tissue repair agents because of their protecting and potentiating effects with heparin binding growth factors. Moreover, dextran derivatives in contact with cells such as endothelial cells, smooth muscle cells, or tumoral cells can affect both cell proliferation and metabolism. It appears that these bioactive polymers are also efficient tools to investigate the precise mechanism of action of individual biological activities by contrasting their mode of action to that of heparin. In addition to their numerous biological properties and biospecificity, functionalized dextrans are relatively simple to manufacture and exempt of donor contaminant, which make them attractive in a variety of clinical applications.