Véronique Viateau

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.

Use of the induced membrane technique for bone tissue engineering purposes: animal studies.

Animal experiments using the induced membrane procedure for bone tissue engineering purposes have provided evidence that the membrane has structural characteristics and biologic properties that may be used for bone tissue engineering purposes. Clinically relevant animal models have demonstrated that standardized particulate bone constructs can be used to repair large bone defects using the procedure and that the osteogenic ability of these constructs partially approaches that of bone autografts.

The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs.

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Indwelling double pigtail ureteral stent combined or not with surgery for feline ureterolithiasis: complications and outcome in 15 cases.

Ureteral obstruction secondary to ureterolithiasis in cats is a challenging situation. Ureteral stenting has recently been introduced to prevent complications that often occurred after ureterotomy or other invasive surgeries. The purpose of this study is to describe the stenting technique and perioperative difficulties, as well as long-term outcome and complications with ureteral stenting in 12 cats with ureteroliths. Fifteen 2.5 Fr soft double pigtail multi-fenestrated ureteral stents were placed in an anterograde fashion under open surgical approaches and with fluoroscopic guidance in 12 cats. Nine cats received a unilateral stent and three received bilateral stents. Ureterotomy or ureteral resection and end-to-end anastomosis were performed in three and four cases, respectively. In six cats, papillotomy was performed to facilitate dilatator and stent placement. All cats recovered well from the surgical procedure, except one cat, which died during the anaesthesia recovery period. Postoperative complications included dysuria (three cases, diagnosed at 15 days, 1 month and 3 months, respectively), urinary tract infection (one case, 1 month after surgery), stent migration requiring stent replacement (one case, 19 months after surgery) and stent obstruction requiring stent removal (three cases with previously end-to-end anastomosis between 2 and 8 months after surgery). Nine cats (75%) were alive at a mean follow-up of 453 ± 194 (123–720) days. The median survival time was >415 days. Stent placement appeared to be a valuable and safe option for treating ureteral obstruction in cats. However, periodic and long-term monitoring of stents is warranted.

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model.

Tissue‐engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold‐material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta‐tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous‐pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta‐tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study. Copyright

The influence of Bone Morphogenic Protein-2 on the consolidation phase in a distraction osteogenesis model

We asked whether locally applied recombinant-Bone Morphogenic Protein-2 (rh-BMP-2) with an absorbable Type I collagen sponge (ACS) carrier could enhance the consolidation phase in a callotasis model. We performed unilateral transverse osteotomy of the tibia in 21 immature male rabbits. After a latency period of 7 days, a 3-weeks distraction was begun at a rate of 0.5mm/12h. At the end of the distraction period (Day 28) animals were randomly divided into three groups and underwent a second surgical procedure: 6 rabbits in Group I (Control group; the callus was exposed and nothing was added), 6 rabbits in Group II (ACS group; receiving the absorbable collagen sponge soaked with saline) and 9 rabbits in Group III (rh-BMP-2/ACS group; receiving the ACS soaked with 100μg/kg of rh-BMP-2, Inductos(®), Medtronic). Starting at Day 28 we assessed quantitative and qualitative radiographic parameters as well as densitometric parameters every two weeks (Days 28, 42, 56, 70 and 84). Animals were sacrificed after 8 weeks of consolidation (Day 84). Qualitative radiographic evaluation revealed hypertrophic calluses in the Group III animals. The rh-BMP-2/ACS also influenced the development of the cortex of the calluses as shown by the modified radiographic patterns in Group III when compared to Groups I and II. Densitometric analysis revealed the bone mineral content (BMC) was significantly higher in the rh-BMP-2/ACS treated animals (Group III).

One Stage Functional End‐to‐End Stapled Intestinal Anastomosis and Resection Performed by Nonexpert Surgeons for the Treatment of Small Intestinal Obstruction in 30 Dogs

OBJECTIVES To describe stapled 1-stage functional end-to-end intestinal anastomosis for treatment of small intestinal obstruction in dogs and evaluate outcome when the technique is performed by nonexpert surgeons after limited training in the technique. STUDY DESIGN Case series. ANIMALS Dogs (n=30) with intestinal lesions requiring an enterectomy. METHODS Stapled 1-stage functional end-to-end anastomosis and resection using a GIA-60 and a TA-55 stapling devices were performed under supervision of senior residents and faculty surgeons by junior surgeons previously trained in the technique on pigs. Procedure duration and technical problems were recorded. Short-term results were collected during hospitalization and at suture removal. Long-term outcome was established by clinical and ultrasonographic examinations at least 2 months after surgery and from written questionnaires, completed by owners. RESULTS Mean±SD procedure duration was 15±12 minutes. Postoperative recovery was uneventful in 25 dogs. One dog had anastomotic leakage, 1 had a localized abscess at the transverse staple line, and 3 dogs developed an incisional abdominal wall abscess. No long-term complications occurred (follow-up, 2-32 months). CONCLUSION Stapled 1-stage functional end-to-end anastomosis and resection is a fast and safe procedure in the hand of nonexpert but trained surgeons.Objectives: To describe stapled 1-stage functional end-to-end intestinal anastomosis for treatment of small intestinal obstruction in dogs and evaluate outcome when the technique is performed by nonexpert surgeons after limited training in the technique. Study Design: Case series. Animals: Dogs (n=30) with intestinal lesions requiring an enterectomy. Methods: Stapled 1-stage functional end-to-end anastomosis and resection using a GIA-60 and a TA-55 stapling devices were performed under supervision of senior residents and faculty surgeons by junior surgeons previously trained in the technique on pigs. Procedure duration and technical problems were recorded. Short-term results were collected during hospitalization and at suture removal. Long-term outcome was established by clinical and ultrasonographic examinations at least 2 months after surgery and from written questionnaires, completed by owners. Results: Mean±SD procedure duration was 15±12 minutes. Postoperative recovery was uneventful in 25 dogs. One dog had anastomotic leakage, 1 had a localized abscess at the transverse staple line, and 3 dogs developed an incisional abdominal wall abscess. No long-term complications occurred (follow-up, 2–32 months). Conclusion: Stapled 1-stage functional end-to-end anastomosis and resection is a fast and safe procedure in the hand of nonexpert but trained surgeons.

Low-dose BMP-2 and MSC dual delivery onto coral scaffold for critical-size bone defect regeneration in sheep

Tissue‐engineered constructs (TECs) combining resorbable calcium‐based scaffolds and mesenchymal stem cells (MSCs) have the capability to regenerate large bone defects. Inconsistent results have, however, been observed, with a lack of osteoinductivity as a possible cause of failure. This study aimed to evaluate the impact of the addition of low‐dose bone morphogenetic protein‐2 (BMP‐2) to MSC‐coral‐TECs on the healing of clinically relevant segmental bone defects in sheep. Coral granules were either seeded with autologous MSCs (bone marrow‐derived) or loaded with BMP‐2. A 25‐mm‐long metatarsal bone defect was created and stabilized with a plate in 18 sheep. Defects were filled with one of the following TECs: (i) BMP (n = 5); (ii) MSC (n = 7); or (iii) MSC‐BMP (n = 6). Radiographic follow‐up was performed until animal sacrifice at 4 months. Bone formation and scaffold resorption were assessed by micro‐CT and histological analysis. Bone union with nearly complete scaffold resorption was observed in 1/5, 2/7, and 3/6 animals, when BMP‐, MSC‐, and MSC‐BMP‐TECs were implanted, respectively. The amount of newly formed bone was not statistically different between groups: 1074 mm3 [970–2478 mm3], 1155 mm3 [970–2595 mm3], and 2343 mm3 [931–3276 mm3] for BMP‐, MSC‐, and MSC‐BMP‐TECs, respectively. Increased scaffold resorption rate using BMP‐TECs was the only potential side effect observed. In conclusion, although the dual delivery of MSCs and BMP‐2 onto a coral scaffold further increased bone formation and bone union when compared to single treatment, results were non‐significant. Only 50% of the defects healed, demonstrating the need for further refinement of this strategy before clinical use.

Bone tissue response induced by bioactive polymer functionalized Ti6Al4V surfaces: In vitro and in vivo study

Ti6Al4V is commonly used for orthopedic applications. This study was designed to test the potentially added benefit of Ti6Al4V functionalized with a bioactive polymer poly(sodium styrene sulfonate) both in vitro and in vivo. Cell-based assays with MC3T3-E1 osteoblast-like cells were used to measure the cell adhesion strength, cell spreading, focal contact formation, cell differentiation and the mineralization of extracellular matrix on grafted and ungrafted Ti6Al4V discs in combination with FBS and collagen type I. Bone morphogenetic protein-2 (BMP-2) was also included in the cell differentiation assay. Results showed that the grafted surface combined with collagen I gave superior levels in every parameter tested with cell-based assays and was almost equivalent to BMP-2 for cell differentiation. In vivo testing was conducted in rabbits (n=42) with cylinders of grafted and ungrafted Ti6Al4V implanted in defects made to the femoral and lateral condyles and animals that were maintained to 1, 3 and 12months. Hydroxyapatite coated Ti6Al4V cylinders were included as a clinical reference control. Osseointegration was assessed post-mortem using histomorphometric analysis conducted on resin sections of explanted undecalcified bone. Two histomorphometric parameters, that of bone-to-implant contact and the bone area, were analyzed by a trained observer blinded to sample identity. Results showed osseointegration on grafted Ti6Al4V was marginally better than both ungrafted and hydroxyapatite coated Ti6Al4V. Overall, the study found that the grafted Ti6Al4V significantly promoted all aspects of osteogenesis tested in vitro and, although in vivo outcomes were less compelling, histomorphometry showed osseointegration of grafted Ti6Al4V implants was equivalent or better than controls.

Coral Scaffolds in Bone Tissue Engineering and Bone Regeneration

Coral exoskeleton, which consists of CaCO3 and has an interconnected-pore structure that resembles that of natural human bone, has been used as scaffold material to fill bone defects in both animal models and humans since the early 1970s. This natural material is biocompatible, osteoconductive, and biodegradable. Most importantly, the possibility of seeding coral scaffolds with either stem cells or loading them with growth factors has provided novel alternatives for bone tissue engineering. In vitro studies have demonstrated that (1) seeded cells adhered and proliferated in a time-dependent manner, and (2) loaded growth factors were absorbed on coral scaffold and subsequently released. Moreover, when coral scaffolds containing either cells and/or growth factors were implanted in vivo, a significantly increased amount of newly-formed bone was observed. Most importantly, timely resorption of the scaffold material in vivo was associated with full bone regeneration in a clinically-relevant sheep model of bone defect. Although sometimes inconsistent, such outcomes provided evidence that bone regeneration, which matches, and even supersedes, the efficacy of autologous bone graft, is achievable with coral scaffolds.