Understanding and leveraging cell metabolism to enhance mesenchymal stem cell transplantation survival in tissue engineering and regenerative medicine applications.

In tissue engineering and regenerative medicine, stem cell-specifically, mesenchymal stromal/stem cells (MSCs)-therapies have fallen short of their initial promise and hype. The observed marginal, to no benefit, success in several applications has been attributed primarily to poor cell survival and engraftment at transplantation sites. MSCs have a metabolism that is flexible enough to enable them to fulfill their various cellular functions and remarkably sensitive to different cellular and environmental cues. At the transplantation sites, MSCs experience hostile environments devoid or, at the very least, severely depleted of oxygen and nutrients. The impact of this particular setting on MSC metabolism ultimately affects their survival and function. In order to develop the next generation of cell-delivery materials and methods, scientists must have a better understanding of the metabolic switches MSCs experience upon transplantation. By designing treatment strategies with cell metabolism in mind, scientists may improve survival and the overall therapeutic potential of MSCs. Here, we provide a comprehensive review of plausible metabolic switches in response to implantation and of the various strategies currently used to leverage MSC metabolism to improve stem cell-based therapeutics.

Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation.

Mesenchymal stem cells (MSCs) hold considerable promise in tissue engineering (TE). However, their poor survival when exogenously administered limits their therapeutic potential. Previous studies from our group demonstrated that lack of glucose (glc) (but not of oxygen) is fatal to human MSCs because it serves as a pro-survival and pro-angiogenic molecule for human MSCs (hMSCs) upon transplantation. However, which energy-providing pathways MSCs use to metabolize glc upon transplantation? Are there alternative energetic nutrients to replace glc? And most importantly, do hMSCs possess significant intracellular glc reserves for ensuring their survival upon transplantation? These remain open questions at the forefront of TE based-therapies. In this study, we established for the first time that the in vivo environment experienced by hMSCs is best reflected by near-anoxia (0.1% O2 ) rather than hypoxia (1%-5% O2 ) in vitro. Under these near-anoxia conditions, hMSCs rely almost exclusively on glc through anerobic glycolysis for ATP production and are unable to use either exogenous glutamine, serine, or pyruvate as energy substrates. Most importantly, hMSCs are unable to adapt their metabolism to the lack of exogenous glc, possess a very limited internal stock of glc and virtually no ATP reserves. This lack of downregulation of energy turnover as a function of exogenous glc level results in a rapid depletion of hMSC energy reserves that explains their poor survival rate. These new insights prompt for the development of glc-releasing scaffolds to overcome this roadblock plaguing the field of TE based-therapies. Stem Cells 2018;36:363-376.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells.

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Quiescence Preconditioned Human Multipotent Stromal Cells Adopt a Metabolic Profile Favorable for Enhanced Survival under Ischemia.

A major impediment to the development of therapies with mesenchymal stem cells/multipotent stromal cells (MSC) is the poor survival and engraftment of MSCs at the site of injury. We hypothesized that lowering the energetic demand of MSCs by driving them into a quiescent state would enhance their survival under ischemic conditions. Human MSCs (hMSCs) were induced into quiescence by serum deprivation (SD) for 48 hours. Such preconditioned cells (SD-hMSCs) exhibited reduced nucleotide and protein syntheses compared to unpreconditioned hMSCs. SD-hMSCs sustained their viability and their ATP levels upon exposure to severe, continuous, near-anoxia (0.1% O2 ) and total glucose depletion for up to 14 consecutive days in vitro, as they maintained their hMSC multipotential capabilities upon reperfusion. Most importantly, SD-hMSCs showed enhanced viability in vivo for the first week postimplantation in mice. Quiescence preconditioning modified the energy-metabolic profile of hMSCs: it suppressed energy-sensing mTOR signaling, stimulated autophagy, promoted a shift in bioenergetic metabolism from oxidative phosphorylation to glycolysis and upregulated the expression of gluconeogenic enzymes, such as PEPCK. Since the presence of pyruvate in cell culture media was critical for SD-hMSC survival under ischemic conditions, we speculate that these cells may utilize some steps of gluconeogenesis to overcome metabolic stress. These findings support that SD preconditioning causes a protective metabolic adaptation that might be taken advantage of to improve hMSC survival in ischemic environments. Stem Cells 2017;35:181-196.

Proangiogenic and prosurvival functions of glucose in human mesenchymal stem cells upon transplantation.

A major limitation in the development of cellular therapies using human mesenchymal stem cells (hMSCs) is cell survival post-transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. We demonstrated that hMSCs could endure sustained near-anoxia conditions only in the presence of glucose. In this in vitro cell model, the protein expressions of Hif-1α and angiogenic factors were upregulated by the presence of glucose. Ectopically implanted tissue constructs supplemented with glucose exhibited four- to fivefold higher viability and were more vascularized compared to those without glucose at day 14. These findings provided the first direct in vitro and in vivo demonstration of the proangiogenic and prosurvival functions of glucose in hMSC upon transplantation and identified glucose as an essential component of the ideal scaffold for transplanting stem cells.

The Lower in Vivo Osteogenicity of Adipose Tissue-Derived Stem Cells Correlates with a Higher Innate Immune Response.

Adipose tissue-derived mesenchymal stem cells (ATSCs) have been used as an alternative to bone marrow-derived mesenchymal stem cells (BMSCs) for bone tissue engineering applications. The ability of ATSCs to promote new bone formation remains lower than that of BMSCs. This study aimed to investigate the mechanisms underlying osteogenicity differences between human ATSCs and BMSCs in ceramic constructs, focusing on the effects of inflammation on this process. In contrast to ATSC-containing constructs, which did not induce bone formation in an ectopic mouse model, BMSC constructs consistently did so. Gene expression analysis revealed that human BMSCs, concomitantly with host murine progenitors, differentiated into the osteogenic lineage early post-implantation. In contrast, ATSCs differentiated later, when few implanted viable cells remained post-implantation, while the host murine cells did not differentiate. Comparison of the inflammatory profile in the cell constructs indicated concomitant upregulation of some human and murine inflammatory genes in the ATSC-constructs compared to the BMSC-constructs during the first-week post-implantation. The high level of chemokine production by the ATSCs was confirmed at the gene and protein levels before implantation. The immune cell recruitment within the constructs was then explored post-implantation. Higher numbers of TRAP-/ MRC1 (CD206) + multinucleated giant cells, NOS2 + M1, and ARG1 + M2 macrophages were present in the ATSC constructs than in the BMSC constructs. These results proved that ATSCs are a transient source of inflammatory cytokines promoting a transient immune response post-implantation; this milieu correlates with impaired osteogenic differentiation of both the implanted ATSCs and the host osteoprogenitor cells.

Efficient autocrine and paracrine signaling explain the osteogenic superiority of transgenic BMP-2 over rhBMP-2.

Bone morphogenetic protein-2 (BMP-2) is an osteogenic protein used clinically to enhance bone healing. However, it must be applied in very high doses, causing adverse side effects and increasing costs while providing only incremental benefit. Preclinical models of bone healing using gene transfer to deliver BMP-2 suggest that transgenic BMP-2 is much more osteogenic than rhBMP-2. Using a reporter mesenchymal cell line, we found transgenic human BMP-2 cDNA to be at least 100-fold more effective than rhBMP-2 in signaling. Moreover, a substantial portion of the BMP-2 produced by the transduced cells remained cell associated. Signaling by transgenic BMP-2 occurred via binding to the type I receptor, activating the associated kinase and generating phospho-smads. Signaling was partially resistant to noggin, an important extracellular inhibitor of BMP-2, possibly because nascent BMP-2 binds to its cell surface receptor during secretion and thus signals in a protected peri-cellular environment. Although the amounts of BMP-2 secreted by the transduced cells were too low to affect distant cells, transduced cells were able to induce signaling in a paracrine fashion that required close proximity of the cells, possibly cell-to-cell contact. The greater osteogenic potency of transgenic BMP-2 was confirmed with human bone marrow stromal cells.

3D-Printed Osteoinductive Polymeric Scaffolds with Optimized Architecture to Repair a Sheep Metatarsal Critical-Size Bone Defect.

The reconstruction of critical-size bone defects in long bones remains a challenge for clinicians. A new osteoinductive medical device is developed here for long bone repair by combining a 3D-printed architectured cylindrical scaffold made of clinical-grade polylactic acid (PLA) with a polyelectrolyte film coating delivering the osteogenic bone morphogenetic protein 2 (BMP-2). This film-coated scaffold is used to repair a sheep metatarsal 25-mm long critical-size bone defect. In vitro and in vivo biocompatibility of the film-coated PLA material is proved according to ISO standards. Scaffold geometry is found to influence BMP-2 incorporation. Bone regeneration is followed using X-ray scans, µCT scans, and histology. It is shown that scaffold internal geometry, notably pore shape, influenced bone regeneration, which is homogenous longitudinally. Scaffolds with cubic pores of ≈870 µm and a low BMP-2 dose of ≈120 µg cm-3 induce the best bone regeneration without any adverse effects. The visual score given by clinicians during animal follow-up is found to be an easy way to predict bone regeneration. This work opens perspectives for a clinical application in personalized bone regeneration.

Enzyme-controlled, nutritive hydrogel for mesenchymal stromal cell survival and paracrine functions.

Culture-adapted human mesenchymal stromal cells (hMSCs) are appealing candidates for regenerative medicine applications. However, these cells implanted in lesions as single cells or tissue constructs encounter an ischemic microenvironment responsible for their massive death post-transplantation, a major roadblock to successful clinical therapies. We hereby propose a paradigm shift for enhancing hMSC survival by designing, developing, and testing an enzyme-controlled, nutritive hydrogel with an inbuilt glucose delivery system for the first time. This hydrogel, composed of fibrin, starch (a polymer of glucose), and amyloglucosidase (AMG, an enzyme that hydrolyze glucose from starch), provides physiological glucose levels to fuel hMSCs via glycolysis. hMSCs loaded in these hydrogels and exposed to near anoxia (0.1% pO2) in vitro exhibited improved cell viability and angioinductive functions for up to 14 days. Most importantly, these nutritive hydrogels promoted hMSC viability and paracrine functions when implanted ectopically. Our findings suggest that local glucose delivery via the proposed nutritive hydrogel can be an efficient approach to improve hMSC-based therapeutic efficacy.

Secondary structure of rhBMP-2 in a protective biopolymeric carrier material.

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular ß-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.

In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes.

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.

Osteogenic-differentiated mesenchymal stem cell-secreted extracellular matrix as a bone morphogenetic protein-2 delivery system for ectopic bone formation.

While human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a "bony matrix" containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier.

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation.

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. STATEMENT OF SIGNIFICANCE: mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.

RNA-based therapy for osteogenesis.

Nucleic acid-based therapy has shown great promise in accelerating bone regeneration as well as other diseases. Nucleic acids used in gene therapy mainly are either plasmid DNA (pDNA) or RNAs. Although pDNA therapy has been extensively studied for decades with encouraging preclinical and clinical results, side effects, and low efficiency associated with nuclear trafficking are hard to bypass. Unlike pDNA, RNAs (mRNA, siRNA, miRNA) exert their function in the cytoplasm, thereby being more efficient in hard-to-transfect cells such as primary osteoblasts. RNA interference-based gene silencing represents a negative regulation which knockdown the expression of antagonists that impair osteogenesis process. In contrary, mRNA therapy for osteogenesis represents a positive regulation which delivers mRNA encoding growth factors to accelerate bone regeneration. This review presents a comprehensive summary of the mRNA and siRNA-based therapies and the targets for bone regeneration in case of bone defect and osteoporosis.

Effect of the Bone Morphogenetic Protein-2 Doses on the Osteogenic Potential of Human Multipotent Stromal Cells- Containing Tissue Engineered Constructs.

IMPACT STATEMENT: A strategy for improving the efficacy of stem cell-based bone tissue engineering (TE) constructs is to combine bone morphogenetic protein-2 (BMP-2) with multipotent stromal cells (MSC). Previous studies on the potential cooperative effect of BMP-2 with human multipotent stromal cells (hMSCs) on bone formation in vivo have, however, shown contradictory results likely due to the various and/or inappropriate BMP-2 doses. Our results provided evidence that the addition of BMP-2 at low dose only was beneficial to improve the osteogenic potential of hMSCs-containing TE constructs, whereas BMP-2 delivered at high dose overcame the advantage of combining this growth factor with hMSCs. This new knowledge will help in designing improved combination strategies for tissue regeneration with better clinical outcomes.

Functionalization of phosphocalcic bioceramics for bone repair applications.

This work is devoted to the processing of bone morphogenetic protein (BMP-2) functionalized silicate substituted hydroxyapatite (SiHA) ceramic spheres. The motivation behind it is to develop injectable hydrogel/bioceramic composites for bone reconstruction applications. SiHA microspheres were shaped by spray drying and thoroughly characterized. The silicate substitution was used to provide preferred chemical sites at the ceramic surface for the covalent immobilization of BMP-2. In order to control the density and the release of the immobilized BMP-2, its grafting was performed via ethoxysilanes and polyethylene glycols. A method based on Kaiser's test was used to quantify the free amino groups of grafted organosilanes available at the ceramic surface for BMP-2 immobilization. The SiHA surface modification was investigated by means of X-ray photoelectron spectroscopy, Fourier transformed infrared spectroscopy and thermogravimetry coupled with mass spectrometry. The BMP-2 bioactivity was assessed, in vitro, by measuring the luciferase expression of a stably transfected C3H10 cell line (C3H10-BRE/Luc cells). The results provided evidence that the BMP-2 grafted onto SiHA spheres remained bioactive.

Osteogenic potential of adipogenic predifferentiated human bone marrow-derived multipotent stromal cells for bone tissue-engineering.

In the present study, we evaluated the benefits of an adipogenic predifferentiation, the pathway most closely related to osteoblastogenesis, on the pro-osteogenic potential of human adult multipotent bone marrow stromal cells (hBMSCs), both in vitro and in vivo. Adipogenic differentiation of hBMSCs for 14 days resulted in a heterogeneous cell population from which the most adipogenic-committed cells were eliminated by their lack of readhesion ability. Our results provided evidence that the select adherent adipogenic differentiated hBMSCs (sAD+ cells) express a gene profile characteristic of both adipogenic and osteogenic lineages. In vitro, when cultured in osteogenic medium, sAD+ differentiated along the osteogenic lineage faster than undifferentiated hBMSCs. In vivo, in an ectopic mouse model, sAD+ exhibited a significantly higher bone formation capability compared with undifferentiated hBMSCs. We sought, then, to investigate the underlying mechanisms responsible for such beneficial effects of adipogenic predifferentiation on bone formation and found that this outcome was not linked to a better cell survival post-implantation. The secretome of sAD+ was both proangiogenic and chemoattractant, but its potential did not supersede the one of undifferentiated hBMSCs. However, using co-culture systems, we observed that the sAD+ paracrine factors were pro-osteogenic on undifferentiated hBMSCs. In conclusion, adipogenic priming endows hBMSCs with high osteogenic potential as well as pro-osteogenic paracrine-mediated activity. This preconditioning appears as a promising strategy for bone tissue engineering technology in order to improve the hBMSC osteogenic potency in vivo.

Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression.

Mesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O(2)) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48 h) exposure to hypoxia (

Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death.

Mesenchymal stem cells (MSCs) have been proposed for the repair of damaged tissue including bone, cartilage, and heart tissue. Upon in vivo transplantation, the MSCs encounter an ischemic microenvironment characterized by reduced oxygen (O2) tension and nutrient deprivation that may jeopardize viability of the tissue construct. The aim of this study was to assess the effects of serum deprivation and hypoxia on the MSC survival rates in vitro. As expanded MSCs are transferred from plastic to a scaffold in most tissue engineering approaches, possibly inducing loss of survival signals from matrix attachments, the effects of a scaffold shift on the MSC survival rates were also assessed. Human MSCs were exposed for 48 hours to (i) a scaffold substrate shift, (ii) serum deprivation, and (iii) O2 deprivation. MSCs were also exposed to prolonged (up to 120 hours) hypoxia associated with serum deprivation. Cell death was assessed by Live/Dead staining and image analysis. The MSC death rates were not affected by the shift to scaffold or 48-hour hypoxia, but increased with fetal bovine serum (FBS) starvation, suggesting that between the two components of ischemia, nutrient deprivation is the stronger factor. Long-term hypoxia combined with serum deprivation resulted in the complete death of MSCs (99 +/- 1%), but this rate was reduced by half when MSCs were exposed to hypoxia in the presence of 10% FBS (51 +/- 31%). These results show that MSCs are sensitive to the concurrent serum and O2 deprivation to which they are exposed when transplanted in vivo, and call for the development of new transplantation methods.

Long-bone critical-size defects treated with tissue-engineered grafts: a study on sheep.

Standardized particulate bone constructs, obtained by expanding autologous mesenchymal stem cells (MSCs) onto coral granules in vitro, were transplanted into long-bone, critical-size defects in sheep. Control experiments were also performed in which autologous bone grafts were implanted. Defect cavities were lined with a preformed vascularized membrane (induced by temporarily inserting a cement spacer for 6 weeks prior to bone construct implantation), which served as a mold keeping the engineered bone granules in place. Radiographic, histological, and computed tomographic tests performed 6 months later showed that the osteogenic abilities of the engineered construct and autograft were significantly greater than those of coral scaffold alone. No significant differences were found between the amount of newly formed bone in defects filled with coral/MSCs and those filled with autograft, yet radiological scores differed significantly between the two groups (21% and 100% healed cortices, respectively). The present study on a clinically relevant animal model provides the first evidence that standardized particulate bone constructs can be used to repair large bone defects and that their osteogenic ability approaches that of bone autograft, the bone repair benchmark. By proving feasibility, the present study makes possible the treatment of segmental bone losses with bone constructs engineered from granules, a process which is much simpler than preparing customized massive constructs using computer-assisted techniques. Important parameters, such as the rate of scaffold resorption and the number of MSCs to be seeded on the scaffolds, need to be optimized before reaching pertinent definitive conclusions.