Pulmonary Embolism after Core Decompression of the Femoral Head Using Injectable Bone Graft Substitute: A Case Report.

The authors report a rare complication regarding the case of an 18-year-old female with bilateral osteonecrosis of the femoral head (ONFH) secondary to the treatment and hematopoietic stem cell transplant (HSCT) of an acute lymphoblastic leukemia (ALL). The patient underwent a bilateral necrotic bone debridement and core decompression (CD) surgery with injectable synthetic bone graft, which unfortunately caused a pulmonary injectable bone graft substitute embolism.

Derivation of Mesenchymal Stromal Cells from Ovine Umbilical Cord Wharton's Jelly.

The methods described herein allow for the isolation and expansion of fibroblastic-like ovine Wharton's jelly-derived mesenchymal stromal cells (oWJ-MSC) that, similarly to their human counterparts, adhere to standard plastic surfaces in culture; show a mesenchymal profile for specific surface antigens (i.e., positive for CD44 and CD166); and lack expression of endothelial (CD31) and hematopoietic (CD45) markers as well as major histocompatibility complex (MHC) class-II. Homogeneous cell cultures result from a two-phase bioprocess design that starts with the isolation of mesenchymal stromal cells (MSC) from the Wharton's jelly of ovine umbilical cords up to a first step of cryopreservation. The second phase allows for further expansion of ovine WJ-MSC up to sufficient numbers for further studies. Overall, this methodology encompasses a 2-week bioprocess design that encompasses two cell culture passages ensuring sufficient cells for the generation of a Master Cell Bank. Further thawing and scale expansion results in large quantities of oWJ-MSC that can be readily used in proof of efficacy and safety studies in the preclinical development stage of the development of cell-based medicines. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation and expansion of ovine mesenchymal stromal cells from Wharton's jelly of the umbilical cord Basic Protocol 2: Characterization of ovine mesenchymal stromal cells Basic Protocol 3: Growth profile determination of ovine mesenchymal stromal cells from Wharton's jelly.

Advances in translational orthopaedic research with species-specific multipotent mesenchymal stromal cells derived from the umbilical cord.

Compliance with current regulations for the development of innovative medicines require the testing of candidate therapies in relevant translational animal models prior to human use. This poses a great challenge when the drug is composed of cells, not only because of the living nature of the active ingredient but also due to its human origin, which can subsequently lead to a xenogeneic response in the animals. Although immunosuppression is a plausible solution, this is not suitable for large animals and may also influence the results of the study by altering mechanisms of action that are, in fact, poorly understood. For this reason, a number of procedures have been developed to isolate homologous species-specific cell types to address preclinical pharmacodynamics, pharmacokinetics and toxicology. In this work, we present and discuss advances in the methodologies for derivation of multipotent Mesenchymal Stromal Cells derived from the umbilical cord, in general, and Wharton's jelly, in particular, from medium to large animals of interest in orthopaedics research, as well as current and potential applications in studies addressing proof of concept and preclinical regulatory aspects.

Effect of Allogeneic Cell-Based Tissue-Engineered Treatments in a Sheep Osteonecrosis Model.

Osteonecrosis of the femoral head (ONFH) is defined as a tissue disorder and successive subchondral bone collapse resulting from an ischemic process, which may progress to hip osteoarthritis. Cell therapy with multipotent bone marrow mesenchymal stromal cells (BM-MSC) of autologous origin appears to be safe and has shown regenerative potential in previous preclinical and clinical studies. The use of allogeneic cells is far more challenging, but may be a promising alternative to use of autologous cells. Moreover, an optimized dosage of cells from an allogeneic source is needed to obtain off-the-shelf tissue engineering products (TEPs). The purpose of this study was to evaluate the efficacy of a TEP composed of undifferentiated ex vivo expanded BM-MSC of allogeneic origin, combined with bone matrix particles in variable doses. A comparative analysis of TEP's bone regenerative properties against its autologous counterpart was performed in an early-stage ONFH preclinical model in mature sheep. Allogeneic BM-MSC groups demonstrated bone regeneration capacity in osteonecrotic lesions equivalent to autologous BM-MSC groups 6 weeks after treatment. Likewise, stimulation of bone regeneration by a low cell dose of 0.5 × 106 BM-MSC/cm3 was equivalent to that of a high cell dose, 5 × 106 BM-MSC/cm3. Neither local nor systemic immunological reactions nor tumorigenesis were reported, strengthening the safety profile of allogeneic BM-MSC therapy in this model. Our results suggest that low-dose allogeneic BM-MSC is sufficient to promote bone regeneration in femoral head osteonecrotic lesions, and should be considered in translation of new allogeneic cell-based TEPs to human clinics. Impact statement Cell therapy and tissue engineering hold promise as novel regenerative therapies for musculoskeletal diseases, and particularly in bone regeneration strategies. In this article, we report the evaluation of the efficacy of an allogeneic cell-based tissue engineering product (TEP) in an early-stage osteonecrosis of the femoral head preclinical model in skeletally mature sheep. Moreover, we demonstrate its bone regeneration capacity and safety in vivo and its equivalence to autologous counterparts. These findings have important implications for the translation of new allogeneic cell-based TEPs to human clinics.

Sterilization Procedure for Temperature-Sensitive Hydrogels Loaded with Silver Nanoparticles for Clinical Applications.

Hydrogels (HG) have recognized benefits as drug delivery platforms for biomedical applications. Their high sensitivity to sterilization processes is however one of the greatest challenges regarding their clinical translation. Concerning infection diseases, prevention of post-operatory related infections is crucial to ensure appropriate patient recovery and good clinical outcomes. Silver nanoparticles (AgNPs) have shown good antimicrobial properties but sustained release at the right place is required. Thus, we produced and characterized thermo-sensitive HG based on Pluronic® F127 loaded with AgNPs (HG-AgNPs) and their integrity and functionality after sterilization by dry-heat and autoclave methods were carefully assessed. The quality attributes of HG-AgNPs were seriously affected by dry-heat methods but not by autoclaving methods, which allowed to ensure the required sterility. Also, direct sterilization of the final HG-AgNPs product proved more effective than of the raw material, allowing simpler production procedures in non-sterile conditions. The mechanical properties were assessed in post mortem rat models and the HG-AgNPs were tested for its antimicrobial properties in vitro using extremely drug-resistant (XDR) clinical strains. The produced HG-AgNPs prove to be versatile, easy produced and cost-effective products, with activity against XDR strains and an adequate gelation time and spreadability features and optimal for in situ biomedical applications.

Vascularized Periosteal Flaps Accelerate Osteointegration and Revascularization of Allografts in Rats.

BACKGROUND: Surgical reconstruction of large bone defects with structural bone allografts can restore bone stock but is associated with complications such as nonunion, fracture, and infection. Vascularized reconstructive techniques may provide an alternative in the repair of critical bone defects; however, no studies specifically addressing the role of vascularized periosteal flaps in stimulating bone allograft revascularization and osseointegration have been reported. QUESTIONS/PURPOSES: (1) Does a vascularized periosteal flap increase the likelihood of union at the allograft-host junction in a critical-size defect femoral model in rats? (2) Does a vascularized periosteal flap promote revascularization of a critical-size defect structural bone allograft in a rat model? (3) What type of ossification occurs in connection with a vascularized periosteal flap? METHODS: Sixty-four rats were assigned to two equal groups. In both the control and experimental groups, a 5-cm critical size femoral defect was created in the left femur and then reconstructed with a cryopreserved structural bone allograft and intramedullary nail. In the experimental group, a vascularized periosteal flap from the medial femoral condyle, with a pedicle based on the descending genicular vessels, was associated with the allograft. The 32 rats of each group were divided into subgroups of 4-week (eight rats), 6-week (eight rats), and 10-week (16 rats) followup. At the end of their assigned followup periods, the animals were euthanized and their femurs were harvested for semiquantitative and quantitative analysis using micro-CT (all followup groups), quantitative biomechanical evaluation (eight rats from each 10-week followup group), qualitative confocal microscopic, backscattered electron microscopic, and histology analysis (4-week and 6-week groups and eight rats from each 10-week followup group). When making their analyses, all the examiners were blinded to the treatment groups from which the samples came. RESULTS: There was an improvement in allograft-host bone union in the 10-week experimental group (odds ratio [OR], 19.29 [3.63-184.50], p < 0.05). In contrast to control specimens, greater bone neoformation in the allograft segment was observed in the experimental group (OR [4-week] 63.3 [39.6-87.0], p < 0.05; OR [6-week] 43.4 [20.5-66.3], p < 0.05; OR [10-week] 62.9 [40.1-85.7], p < 0.05). In our biomechanical testing, control samples were not evaluable as a result of premature breakage during the embedding and assembly processes. Therefore, experimental samples were compared with untreated contralateral femurs. No difference in torsion resistance pattern was observed between both groups. Both backscattered electron microscopy and histology showed newly formed bone tissue and osteoclast lacunae, indicating a regulated process of bone regeneration of the initial allograft in evaluated samples from the experimental group. They also showed intramembranous ossification produced by the vascularized periosteal flap in evaluated samples from the experimental group, whereas samples from the control group showed an attempted endochondral ossification in the allograft-host bone junctions. CONCLUSIONS: A vascularized periosteal flap promotes and accelerates allograft-host bone union and revascularization of cryopreserved structural bone allografts through intramembranous ossification in a preclinical rat model. CLINICAL RELEVANCE: If large-animal models substantiate the findings made here, this approach might be used in allograft reconstructions for critical defects using fibular or tibial periosteal flaps as previously described.

Clinical translation of a mesenchymal stromal cell-based therapy developed in a large animal model and two case studies of the treatment of atrophic pseudoarthrosis.

Pseudoarthrosis is a relatively frequent complication of fractures, in which the lack of mechanical stability and biological stimuli results in the failure of bone union, most frequently in humerus and tibia. Treatment of recalcitrant pseudoarthrosis relies on the achievement of satisfactory mechanical stability combined with adequate local biology. Herein we present two cases of atrophic pseudoarthrosis that received a tissue-engineering product (TEP) composed of autologous bone marrow-derived mesenchymal stromal cells (BM-MSC) combined with deantigenized trabecular bone particles from a tissue bank. The feasibility of the treatment and osteogenic potential of the cell-based medicine was first demonstrated in an ovine model of critical size segmental tibial defect. Clinical-grade autologous BM-MSC were produced following a good manufacturing practice-compliant bioprocess. Results were successful in one case, with pseudoarthrosis resolution, and inconclusive in the other one. The first patient presented atrophic pseudoarthrosis of the humeral diaphysis and was treated with osteosynthesis and TEP resulting in satisfactory consolidation at month 6. The second case presented a recalcitrant pseudoarthrosis of the proximal tibia and the Masquelet technique was followed before filling the defect with the TEP. This patient presented a neuropathic pain syndrome unrelated to the treatment that forced the amputation of the extremity 3 months later. In this case, the histological analysis of the tissue formed at the defect site provided evidence of neovascularization but no overt bone remodelling activity. It is concluded that the use of expanded autologous BM-MSC to treat pseudoarthrosis was demonstrated to be feasible and safe, provided that no clinical complications were reported, and early signs of effectiveness were observed. Copyright © 2016 John Wiley & Sons, Ltd.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 104 MSCs/cm2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

Bernese Periacetabular Osteotomy in a Hip Extra-Articular Resection Followed by Reconstruction Using an Extracorporeal Irradiated Acetabulum Autograft with Megaprosthesis, for Proximal Femur Osteosarcoma in a Pediatric Patient.

Osteosarcoma is the most common primary malignant tumour of bone. The oncologic surgery of a proximal femur osteosarcoma affecting the hip joint can be very challenging. We present an 8-year-old boy with a 5-month history of right hip pain. Radiographs and magnetic resonance imaging (MRI) showed a lytic lesion of the proximal femur extending 13 cm to the diaphysis. Histological evaluation was consistent with high-grade osteoblastic osteosarcoma. After completing chemotherapy we performed an extra-articular resection of the hip. Reconstruction was accomplished by reimplanting the acetabulum after irradiation and modular proximal femur megaprosthesis. Endoprosthetic reconstruction following proximal femur resection is a good treatment alternative achieving good postoperative function. Extra-articular resection of the hip using a periacetabular osteotomy technique enabled us to achieve wide margins and leave an intact posterior column to optimize acetabular reconstruction stability. Extracorporeal irradiation and reimplantation is a valuable treatment option in a situation such as this where allograft geometric fit is a priority. We conclude that an extra-articular resection of the hip, followed by reconstruction with an extracorporeally irradiated acetabulum and a proximal femur modular megaprosthesis, is a useful combined treatment option for malignant lesions involving the hip joint, especially in paediatric patients.

Treatment of femoral head osteonecrosis with advanced cell therapy in sheep.

BACKGROUND: The purpose of this study was to evaluate the efficacy of core decompression associated with advanced cell therapy for the treatment of femoral head osteonecrosis in an established sheep model. METHODS: Early stage osteonecrosis of the right hip was induced cryogenically in 15 mature sheep. At 6 weeks, the sheep were divided into three groups, Group A: core decompression only; Group B: core decompression followed by implantation of an acellular bone matrix scaffold; Group C: core decompression followed by implantation of a cultured BMSC loaded bone matrix scaffold. At 12 weeks, MRI hip studies were performed and then the proximal femur was harvested for histological analysis. RESULTS: In the group of advanced cell therapy, Group C, there was a tendency to higher values of the relative surface of newly formed bone with a mean of 20.3 versus 11.27 % in Group A and 13.04 % in Group B but it was not statistically significant. However, the mean relative volume of immature osteoid was 8.6 % in Group A, 14.97 in Group B, and 53.49 % in Group C (p < 0.05), revealing a greater capacity of osteoid production in the sheep treated with BMSCs. MRI findings were not conclusive due to constant bone edema artifact in all cases. CONCLUSIONS: Our findings indicate that a BMCSs loaded bone matrix scaffold is capable of stimulating bone regeneration more effectively than isolated core decompression or in association with an acellular scaffold in a preclinical femoral head osteonecrosis model in sheep.

Fetoscopic coverage of experimental myelomeningocele in sheep using a patch with surgical sealant.

OBJECTIVE: Assess the feasibility of a fetoscopic patch coverage method for myelomeningocele repair in a sheep model. STUDY DESIGN: Experimental study. A myelomeningocele-like defect was created in 15 fetal sheep on day 75 of gestation. Six remained untreated, whereas 9 underwent fetoscopic coverage of the defect on day 95 of gestation using an inert patch secured with surgical sealant. Clinical and histological examinations were performed after delivery. RESULTS: Four valid newborn lambs were obtained in each group. Mean fetoscopic surgical time was 26.9 (SD=7.4)min. All untreated animals had an open lumbar defect with cerebrospinal fluid leakage, paraplegia, urinary incontinence, and Chiari malformation. All treated animals had a closed defect and were able to walk; one had weak bladder control, and another mild Chiari malformation. CONCLUSION: In a chronic myelomeningocele model in fetal sheep, fetoscopic repair using a sealed patch results in simple, fast, satisfactory neural tube closure and averts neurological damage and Chiari malformation.

A new preclinical femoral head osteonecrosis model in sheep.

INTRODUCTION: Animal models have been used as insight into the pathogenesis of osteonecrosis, even though most have failed to reproduce all stages of human disease, limiting progression in experimental treatment modalities. A new surgically induced animal model of femoral head osteonecrosis in sheep is presented. METHOD: Osteonecrosis was achieved using an improved method of intracephalic cryogenic lesion by means of a cryoprobe and vascular ligation. RESULTS: Histologic findings at 6 and 12 weeks showed progression to advanced stage osteonecrosis. MRI, the gold standard in diagnosis and follow-up in human osteonecrosis showed concordant results with histology. CONCLUSION: Our model can therefore be used as a preclinical femoral head osteonecrosis model in an easily accessible animal to assess biological treatments with MRI.

Inert patch with bioadhesive for gentle fetal surgery of myelomeningocele in a sheep model.

OBJECTIVE: Current techniques used in foetal myelomeningocele repair can require considerable manipulation of fragile foetal tissues to obtain tension-free closure. The aim of this study was to assess the feasibility of a simple foetal coverage method without foetal tissue manipulation to provide closure of the neural tube defect in myelomeningocele. STUDY DESIGN: This is an experimental study performed in 15 foetal sheep with lumbar myelomeningocele, surgically created on day 75 of gestation. Five foetuses remained untreated. Ten underwent coverage with inert sheeting (5 Silastic; 5 Silastic+Marlex) secured by surgical tissue adhesive without suturing on day 95; none of them underwent foetal muscle or skin manipulation. Clinical and subsequent histological examinations were performed at 48h after birth. The Chi-square, Fisher exact, and Mann-Whitney U tests, when appropriate, were used for the comparisons. RESULTS: The mean operating time for foetal coverage was 7.1 (SD=1.6)min. All untreated animals were unable to walk, had sphincter incontinence, showed an open defect, histological spinal cord damage, and a large Chiari malformation. All covered animals were able to walk, had sphincter continence, showed almost complete closure of the defect with regeneration of several soft tissue layers, and minimum Chiari malformation. CONCLUSION: In a surgical myelomeningocele model in sheep, a simple, fast and gentle coverage method using a sealed patch avoids foetal tissue manipulation and enables adequate closure of the neural tube defect, providing regeneration of several tissue layers that protect the spinal cord, and significantly reducing Chiari II malformation.

Fetoscopic release of extremity amniotic bands with risk of amputation.

With the advent of less-invasive fetal surgery techniques, nonlethal disorders are considered amenable to intrauterine treatment. Extremity amniotic band syndrome fulfils the criteria of intrauterine disorders conformable with fetal treatment: capability of prenatal diagnosis and severity. We report 2 cases of extremity amniotic bands with risk of limb amputation released fetoscopically with YAG laser.

Fetal surgery of extremity amniotic bands: an experimental model of in utero limb salvage in fetal lamb.

BACKGROUND: Extremity amniotic band (EAB) syndrome can cause an intrauterine amputation as a result of a mechanical effect with progressive strangulation. The aim of the study is to assess the use of fetal surgery of EABs with risk of amputation in the ovine fetus. METHODS: Right limbs of fifteen 60-day-gestational age fetal sheep were ligated with silk suture at the infracondylar level. Left limbs were used for paired comparison. Fetuses were randomized into 3 groups: an early-repair group (n = 5), a late-repair group (n = 5), and a nonrepaired group (n = 5). The limbs of the 2 repaired groups underwent fetal release. The limbs obtained from at-term fetuses were analyzed morphologically, functionally, and radiologically. Statistical analysis with paired test was used to compare data. RESULTS: Nonrepaired limbs showed amputation or quasi amputation; the repaired ones did not. However, those late-repaired had significantly reduced passive ankle range of motion, shorter limb length, and mild residual changes. CONCLUSIONS: Intrauterine release of potentially severe EABs prevents limb amputation and leads to morphofunctional recovery. Early release shows better results. CLINICAL RELEVANCE: : Intrauterine amputations caused by EABs in the human fetus might be avoidable by fetal surgery.

The effect of prenatal treatment with steroids and preterm delivery in a model of myelomeningocele on the rabbit foetus.

Damage of neural elements (spinal cord and encephalus) in myelomeningocele (MMC) seems to be progressive during gestation because of amniotic fluid chemical contact and continuous leakage of CSF. We studied the effect of preterm delivery and steroid treatment in a model of MMC in the rabbit foetus. Twelve New Zealand White rabbits underwent laparotomy and hysterotomy at 23 days of gestation. Fifty-nine out of 107 foetuses underwent lumbar laminectomy (three to four levels). Dura was opened to expose the neural elements to the amniotic fluid. Six rabbits underwent caesarean section on gestational day 31 for fetal harvest; three of them had no treatment (group T) and three received corticosteroid treatment (group TC). The other six rabbits underwent caesarean section on gestational day 29 for fetal harvest (preterm delivery); three of them had no treatment (group P) and three received corticosteroid treatment (group PC). Alive newborns were clinically, neurophysiologically and histologically analysed. None of mothers died during the procedure. After birth, animals in group preterm showed statistically significant less deformity than animals in group at term. Lower kyphosis was observed in group PC (preterm and steroids). Pain related and spontaneous mobility of lower extremities was higher in groups treated with corticosteroids (TC and PC). Only newborns at term (T and TC groups) showed response to evoked potentials (CMEPs). The response was earlier and higher in group treated with steroids (TC). Histologically, we observed progressive lesion of the spinal cord. Groups treated with steroids (TC and PC) show less inflammatory response. Arnold-Chiari malformation was present in all groups. Animals in group preterm with steroids show statistically significant less herniation than those group at term. Preterm delivery and prenatal steroid therapy seem to be an effective treatment to get less neural injury (spinal cord and encephalus) in myelomeningocele foetuses.