Samer Srouji

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting.

Maxillary sinus membrane lifting is a common procedure aimed at increasing the volume of the maxillary sinus osseous floor prior to inserting dental implants. Clinical observations of bone formation in sinus lifting procedures without grafting bone substitutes were observed, but the biological nature of bone regeneration in sinus lifting procedures is unclear. This study tested whether this osteogenic activity relies on inherent osteogenic capacity residing in the sinus membrane by simulating the in vivo clinical condition of sinus lifting in an animal model. Maxillary sinus membrane cells were cultured in alpha-MEM medium containing osteogenic supplements (ascorbic acid, dexamethasone). Cultured cells revealed alkaline phosphatase activity and mRNA expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and osteonectin) verifying the osteogenic potential of the cells. Fresh tissue samples demonstrated positive alkaline phosphatase enzyme activity situated along the membrane-bone interface periosteum-like layer. To simulate the in vivo clinical conditions, the membranes were folded to form a pocket-like structure and were transplanted subcutaneously in immunodeficient mice for 8 weeks. New bone formation was observed in the transplants indicating the innate osteogenic potential within the maxillary Schneiderian sinus membrane and its possible contribution to bone regeneration in sinus lifting procedures.

Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix

Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

Chitosan–poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model

Tissue engineering sustains the need of a three‐dimensional (3D) scaffold to promote the regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population, allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our group evidenced significant growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded and cultured on melt‐based porous chitosan fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of these in vitro 3D cell constructs. In this study, chitosan‐based scaffolds were seeded and cultured in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in osteogenic culture conditions was performed into critical cranial size defects in nude mice. The objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks of implantation, scaffolds were harvested and prepared for micro‐computed tomography (µCT) analysis. Retrieved implants showed good integration with the surrounding tissue and significant bone formation, more evident for the scaffolds cultured and implanted with human cells. The results of this work demonstrated that chitosan‐based scaffolds, besides supporting in vitro proliferation and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects for their use in bone tissue‐engineering therapies. Copyright

Cell-scaffold transplant of hydrogel seeded with rat bone marrow progenitors for bone regeneration

Bone is the second most frequently transplanted tissue in humans and efforts are focused on developing cell-scaffold constructs which can be employed for autologous implantation in place of allogenic transplants. The objective of the present study was to examine the efficacy of a gelatin-based hydrogel scaffold to support osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) and its application in a cranial defect model. MSCs which were cultured on hydrogel under osteogenic conditions demonstrated typical osteogenic differentiation which included cluster formation with positive Alizarin Red S staining, sedimentation of calcium phosphate as defined by SEM and EDS spectroscopy and expression of mRNA osteogenic markers. Empty scaffolds or those containing either differentiated cells or naïve cells were implanted into cranial defects of athymic nude mice and the healing process was followed by μCT. Substantial bone formation (65%) was observed with osteogenic cell-scaffold constructs when compared to the naïve cell construct (25%) and the cell free scaffold (10%). Results demonstrated the potential of hydrogel scaffolds to serve as a supportive carrier for bone marrow-derived MSCs.

Distraction osteogenesis for tracheostomy dependent children with severe micrognathia.

AbstractObstructive sleep apnea (OSA) in pediatric populations is often associated with congenital craniofacial malformations resulting in decreased pharyngeal airway, which in severe cases leads to tracheostomy dependence. The purpose of this study was to use distraction osteogenesis to improve the airway and decannulate the tracheostomy. This study involved 11 OSA tracheostomy-dependent patients (age range, 4 months to 6 years) who underwent bilateral distraction in the mandibular body using extraoral distraction devices. Following a latency period of 4 days, gradual distraction at a rate of 1 mm/d was performed followed by a consolidation period of 10 weeks. Three-dimensional computed tomography reconstruction of the face and neck before and after the mandibular lengthening aided in quantitative volumetric evaluation of mandibular volume and airway volume. The results demonstrated mandibular elongation of a mean of 30 mm on each side, an increase in mandibular volume by an average of 29.19%, and increase in pharyngeal airway by an average of 70.53%. Two to 3 months following the last lengthening, all 11 patients were decannulated with improvement of signs and symptoms of OSA and elimination of oxygen requirement. Mean follow-up was 2.0 years. The oxygen saturation level rose to more than 95%, and the apnea index respiratory disturbance index was less than 2 episodes per hour for all patients. Bilateral mandibular distraction is a useful method in younger children to decannulate permanent tracheostomy expanding the hypoplastic mandible and concomitantly advance the base of tongue and hyoid bone increasing the pharyngeal airway.

A tissue‐like construct of human bone marrow MSCs composite scaffold support in vivo ectopic bone formation

Biocompatible and osteoconductive cell–scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell–scaffold construct composed of gelatin‐based hydrogel and ceramic (CaCO3/β‐TCP) particles loaded with human MSCs producing a tissue‐like construct applied as a transplant for in vivo bone formation. Bone marrow‐derived human MSCs were cultured in osteogenic induction medium. 5 × 105 (P2) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue‐like construct, shown by histology to contain elongated spindle‐like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue‐like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell‐construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science. Copyright

Lentiviral-Mediated Integrin α5 Expression in Human Adult Mesenchymal Stromal Cells Promotes Bone Repair in Mouse Cranial and Long-Bone Defects

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

Evaluation of the osteoconductive potential of bone substitutes embedded with schneiderian membrane‐ or maxillary bone marrow‐derived osteoprogenitor cells

AIM Sinus augmentation procedures commonly employ osteoconductive scaffolding materials to stimulate and support bone formation. The aim of this study was to develop a simple screening methodology for the evaluation of the osteoconductive potential of various bone graft materials prior to clinical use. MATERIALS AND METHODS Materials tested were Bio-Oss, Bi-Ostetic, OraGraft, and ProOsteon. These Simple and composite bone substitutes were embedded with osteoprogenitor cells derived from either the human maxillary sinus schneiderian membrane (hMSSM) or from maxillary tuberosity bone marrow and then monitored both in vitro and in vivo. RESULTS Cell adherence and proliferation was most pronounced in OraGraft, followed by ProOsteon. In vivo bone formation, within the bone graft, was also observed, with most marked results in OraGraft and ProOsteon grafts. CONCLUSIONS The proposed osteoconductivity testing method proved simple, informative, and reliable for the purpose of screening candidate biomaterials for sinus lifting or sinus augmentation.

Arthroscopic discopexy is effective in managing temporomandibular joint internal derangement in patients with Wilkes stage II and III.

PURPOSE Disc repositioning for temporomandibular joint (TMJ) internal derangement (ID) is a well-established surgical technique with variable success. The purpose of the present study was to assess the outcomes after arthroscopic disc repositioning (discopexy) for TMJ ID. PATIENTS AND METHODS This was a prospective, cohort, single-institutional clinical study. The study included patients with TMJ ID in whom diagnostic arthroscopy had failed. These patients were presented and treated at Miami Oral and Maxillofacial Surgery, Baptist Hospital (Miami FL). The predictive variable was the Wilkes diagnostic categories, presented in 2 groups: II and III versus IV and V. The primary outcome variable was the absence of joint pain at 12 months postoperatively. The secondary outcome variables included joint function, maximum interincisal opening, medication use, joint loading sign, and muscle pain. The patients were followed for 1 year postoperatively. The statistical analyses included paired and independent sample Students t test, χ(2) test, and logistic regression analysis. RESULTS A total of 32 subjects (42 joints), with a mean age of 31 years, were included in the present study; 28 (87.5%) were women. Of the 42 joints, 71.4% were classified as Wilkes stage II and III. A successful outcome was seen in 69% of the studied subjects and in 86.7% of the Wilkes II and III group versus 25% of the Wilkes IV and V group (P = .001). CONCLUSION The results of the present study have shown that TMJ arthroscopic discopexy is an effective and predictable treatment of patients with TMJ ID in whom primary TMJ arthroscopy failed. Our results have also shown that patients with Wilkes II or III TMD will have the most successful outcome.