Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors

Recently, the cannabinoid receptors CB1 and CB2 were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB1 and CB2 receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB2 agonists BML190 and JWH015. The CB1-specific agonist ACEA had no effect, whereas the CB2 antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB2 receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB1 and CB2 receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB2 receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.

Ectopic ossification presenting as osteoid metaplasia in a salivary mucocele in a Shih Tzu dog

Background: Salivary mucocele is an accumulation of saliva in a single or multiloculated cavity lined by connective tissue that is contiguous to a salivary gland-duct complex and is the most common condition affecting the salivary glands in dogs. Occasionally, different types of metaplastic lesions, such as squamous and osseous metaplasia - which are rare lesions in animals - can be observed in association with salivary mucocele.Case presentation: A right facial enlargement was suddenly observed in a 4-year-old non-spayed female Shih-Tzu dog. The lesion presented itself as a soft and fluctuant mass located in the right side of the face near to the neck. Histologically, the mass consisted of a cavitary formation without an epithelial lining. Additionally, microscopic examination revealed the presence of osteoid-producing cells which gave rise to areas of bone formation, probably induced by irritation due to the presence sialoliths. Such cells and bone formations were also present in the cavity wall, consequently leading us to classify the condition as a salivary mucocele with osseous metaplasia.Conclusions: In the present case, the pathogenesis was probably associated with the presence of sialoliths, which can behave as etiological agents for the metaplastic lesion. The occurrence of osteoid metaplasia is a rare peculiar condition in the canine salivar y gland, and due to the rarity and lack of information about this specific disease, no clinical data can yet be associated with the development of salivary mucocele with osseous metaplasia in dogs.

A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the [alpha]2[beta]1 integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

Engineering tubular bone constructs

Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80–120 mm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8- week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (554mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10104mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Physiological investigation of periosteum structure and its application in periosteum tissue engineering

Although many different materials, techniques and methods, including artificial or engineered bone substitutes, have been used to repair various bone defects, the restoration of critical-sized bone defects caused by trauma, surgery or congenital malformation is still a great challenge to orthopedic surgeons. One important fact that has been neglected in the pursuit of resolutions for large bone defect healing is that most physiological bone defect healing needs the periosteum and stripping off the periosteum may result in non-union or non-healed bone defects. Periosteum plays very important roles not only in bone development but also in bone defect healing. The purpose of this project was to construct a functional periosteum in vitro using a single stem cell source and then test its ability to aid the repair of critical-sized bone defect in animal models. This project was designed with three separate but closely-linked parts which in the end led to four independent papers. The first part of this study investigated the structural and cellular features in periostea from diaphyseal and metaphyseal bone surfaces in rats of different ages or with osteoporosis. Histological and immunohistological methods were used in this part of the study. Results revealed that the structure and cell populations in periosteum are both age-related and site-specific. The diaphyseal periosteum showed age-related degeneration, whereas the metaphyseal periosteum is more destructive in older aged rats. The periosteum from osteoporotic bones differs from normal bones both in terms of structure and cell populations. This is especially evident in the cambial layer of the metaphyseal area. Bone resorption appears to be more active in the periosteum from osteoporotic bones, whereas bone formation activity is comparable between the osteoporotic and normal bone. The dysregulation of bone resorption and formation in the periosteum may also be the effect of the interaction between various neural pathways and the cell populations residing within it. One of the most important aspects in periosteum engineering is how to introduce new blood vessels into the engineered periosteum to help form vascularized bone tissues in bone defect areas. The second part of this study was designed to investigate the possibility of differentiating bone marrow stromal cells (BMSCs) into the endothelial cells and using them to construct vascularized periosteum. The endothelial cell differentiation of BMSCs was induced in pro-angiogenic media under both normoxia and CoCl2 (hypoxia-mimicking agent)-induced hypoxia conditions. The VEGF/PEDF expression pattern, endothelial cell specific marker expression, in vitro and in vivo vascularization ability of BMSCs cultured in different situations were assessed. Results revealed that BMSCs most likely cannot be differentiated into endothelial cells through the application of pro-angiogenic growth factors or by culturing under CoCl2-induced hypoxic conditions. However, they may be involved in angiogenesis as regulators under both normoxia and hypoxia conditions. Two major angiogenesis-related growth factors, VEGF (pro-angiogenic) and PEDF (anti-angiogenic) were found to have altered their expressions in accordance with the extracellular environment. BMSCs treated with the hypoxia-mimicking agent CoCl2 expressed more VEGF and less PEDF and enhanced the vascularization of subcutaneous implants in vivo. Based on the findings of the second part, the CoCl2 pre-treated BMSCs were used to construct periosteum, and the in vivo vascularization and osteogenesis of the constructed periosteum were assessed in the third part of this project. The findings of the third part revealed that BMSCs pre-treated with CoCl2 could enhance both ectopic and orthotopic osteogenesis of BMSCs-derived osteoblasts and vascularization at the early osteogenic stage, and the endothelial cells (HUVECs), which were used as positive control, were only capable of promoting osteogenesis after four-weeks. The subcutaneous area of the mouse is most likely inappropriate for assessing new bone formation on collagen scaffolds. This study demonstrated the potential application of CoCl2 pre-treated BMSCs in the tissue engineering not only for periosteum but also bone or other vascularized tissues. In summary, the structure and cell populations in periosteum are age-related, site-specific and closely linked with bone health status. BMSCs as a stem cell source for periosteum engineering are not endothelial cell progenitors but regulators, and CoCl2-treated BMSCs expressed more VEGF and less PEDF. These CoCl2-treated BMSCs enhanced both vascularization and osteogenesis in constructed periosteum transplanted in vivo.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Limitations and challenges in MSC delivery for bone regeneration

Objective: Regeneration of osseous defects by tissue-engineering or cell delivery approach provides a novel means of treatment utilizing cell biology, materials sciences, and molecular biology. The concept of in vitro explanted mesenchymal stem cells (MSCs) with an ability to induce new bone formation has been demonstrated in some small animal models. However, contradictory results have been reported regarding the regenerative capacity of MSCs after ex vivo expansion due to the lack of the understanding of microenvironment for MSC differentiation in vivo. ----- ----- Methods: In our laboratory tissue-derived and bone marrow-derived MSCs have been investigated in their osteogenesis. Cell morphology and proliferation were studied by microscopy, confocal microscopy, FACS and cell counting. Cell differentiation and matrix formation were analysed by matrix staining, quantitative PCR, and immunohistochemistry. A SCID skull defect model was used for cell transplantation studies.----- ----- Results: It was noted that tissue-derived and bone marrow-derived MSCs showed similar characteristics in cell surface marker expression, mesenchymal lineage differentiation potential, and cell population doubling. MSCs from both sources could initiate new bone formation in bone defects after delivery into a critical size defects. The bone forming cells were from both transplanted cells and endogenous cells from the host. Interestingly, the majority of in vitro osteogenic differentiated cells did not form new bone directly even though mineralized matrix was synthesized in vitro by MSCs. Furthermore, no new bone formation was detected when MSCs were transplanted subcutaneously.----- ----- Conclusion: This study unveiled the limitations of MSC delivery in bone regeneration and proposed that in vivo microenvironment needs to be optimized for MSC delivery in osteogenesis.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration