Objectives. The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model. Methods. Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young’s modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey’s post hoc multiple-comparison test. Results. We observed no significant difference in cross-sectional area or in Young’s modulus among the four study groups. In addition, histological sections showed that the BMSCs were aligned well and viable on the tendon slices after two-week culture in groups three and four. Expression levels of several extracellular matrix tendon growth factors, including collagen type I, collagen type III, and matrix metalloproteinase were significantly higher in group four than in group three (p < 0.05). Conclusion. Lateral slits introduced into de-cellularised tendon is a promising method of delivery of BMSCs without compromising cell viability and tendon mechanical properties. In addition, mechanical stimulation of a cell-seeded tendon can promote cell proliferation and enhance expression of collagen types I and III in vitro. Cite this article: J. H. Wu, A. R. Thoreson, A. Gingery, K. N. An, S. L. Moran, P. C. Amadio, C. Zhao. The revitalisation of flexor tendon allografts with bone marrow stromal cells and mechanical stimulation: An ex vivo model revitalising flexor tendon allografts. Bone Joint Res 2017;6:179–185. DOI: 10.1302/2046-3758.63.BJR-2016-0207.R1

Aims. The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. Methods. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21

Aims. Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells. Methods. Cell viability, proliferation, migration, and expression levels of Collagen-I (COL-I) A1, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) were compared between ACL remnant cells untreated and treated with ESW (0.15 mJ/mm. 2. , 1,000 impulses, 4 Hz). To evaluate the subsequent effects on the surrounding cells, bone marrow stromal cells (BMSCs)’ viability, proliferation, migration, and levels of Type I Collagen, Type III Collagen, and tenogenic gene (Scx, TNC) expression were investigated using coculture system. Results. ESW-treated ACL remnant cells presented higher cell viability, proliferation, migration, and increased expression of COL-I A1, TGF-β, and VEGF. BMSC proliferation and migration rate significantly increased after coculture with ACL remnant cells with and without ESW stimulation compared to the BMSCs alone group. Furthermore, ESW significantly enhanced ACL remnant cells’ capability to upregulate the collagen gene expression and tenogenic differentiation of BMSCs, without affecting cell viability, TGF-β, and VEGF expression. Conclusion. ACL remnant cells modulated activity and differentiation of surrounding cells. The results indicated that ESW enhanced ACL remnant cells viability, proliferation, migration, and expression of collagen, TGF-β, VEGF, and paracrine regulation of BMSC proliferation, migration, collagen expression, and tenogenesis. Cite this article: Bone Joint Res 2020;9(8):457–467

Regenerative medicine provides the hope for many intractable diseases as a treatment option and the area is currently the subject of intense investigation in academia and industry. Human bone marrow stromal cells (HBMSCs) possess the ability to differentiate into a variety of cell types of the stromal lineage including cells of the osteogenic and chondrogenic lineages. However, the process of in vitro differentiation is usually inefficient, difficult to reproduce in many cases and, to date, unable to produce homogenous cell populations, which is critical for tissue engineering. Epigenetic regulation of gene expression is recognized as a key mechanism governing cell determination, commitment, and differentiation as well as maintenance of those states. The main components of epigenetic control are DNA methylation and histone acetylation. During development, the epigenetic status changes as cells differentiate along specific lineages. We reasoned that epigenetic modifiers might direct the differentiation pathway of HBMSCs towards either osteogenic or chondrogenic lineage. HBMSCs were serum-starved for 24 hours to synchronise the cell cycle, then treated on three consecutive days either with the DNA demethylating agent 5-Aza-deoxycytidine (5-Aza-dC) 1?M, or the histone deacetylase inhibitor Trichostatin A (TSA) 100 nM or a combination of both. After confluency, the cells were grown in pellet culture for 21 days to facilitate formation of an extracellular matrix. 5-Aza-dC increased the amount of osteoid in the pellet by at least 5 fold compared with controls as assessed by histochemistry, whereas TSA enhanced formation of a cartilage matrix. The differentiation was further enhanced by culturing the pellets in osteogenic or chondrogenic media. These studies suggest that loss of DNA methylation stimulates osteogenic differentiation, whereas inhibition of histone deacetylation favours chondrogenesis. Epigenetic changes thus play an important role in HBMSCs differentiation and offer new approaches in skeletal tissue engineering programs. The challenge will be to define the crucial genes in which loss of DNA methylation has taken place or how changes in histone acetylation (and other histone modifications) affect lineage differentiation

Abstract

Introduction: There is controversy regarding the effectiveness of PRC for bone healing. A possible explanation is the different bone graft substitutes (BGSs) used with PRC. Here we investigated the effect of combining different BGSs with PRC on hBMSCs differentiation and growth factor release from the BGS/PRC composites.

Method: hBMSCs, DBM and allograft were prepared from femoral heads donated by patients undergoing total hip replacement. Growth factor release (TGF-â, VEGF, PDGF-AB, BMP-2) was measured by ELISA. The effect of PRC on hBMSC differentiation was determined by ALP activity and mineralisation. PRC was produced using the CAPTION device (S& N) from 10 healthy volunteers.

Results: Combining PRC with BGSs increased hBMSC proliferation (p< 0.05) and decreased ALP activity (p< 0.05) compared to DBM or â-TCP (GenOS, S& N) alone, but had no effect on allograft following 3 and 5 days treatment. After 21 days PRC enhanced mineralisation compared to all BGSs alone (16%–56%). Compared to PRC alone addition of DBM and allograft increased proliferation (p< 0.05), decreased ALP activity (p< 0.005) and decreased mineralisation (p< 0.005). TGF-â, VEGF and BMP-2 release from PRC was unaffected when combined with DBM but PDGF-AB release was reduced by 50%.

Conclusions: Combining PRC with the majority of BGSs enhanced cell proliferation and decreased osteoblastic differentiation at early time points but increased total mineralisation compared to the BGSs alone. However, compared to PRC alone combining DBM or allograft with PRC reduced mineralisation. One potential explanation for the effects of combining PRC with DBM is altered growth factor release profiles compared to the components alone.

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies. Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

Introduction: Alcohol can induce adipogenesis by bone marrow stromal cells and may cause osteonecrosis of the femoral heads. Currently, there are no medications available to prevent alcohol-induced osteonecrosis. The purpose of this study was to evaluate the effects of puerarin on adipocytic differentiation of bone marrow stromal cells and on the prevention of alcohol-induced osteonecrosis. Materials and Methods: In the in vitro study, bone marrow stromal cells were treated with ethanol as model groups, with ethanol and puerarin as experimental groups, and without ethanol or puerarin to serve as controls. In the in vivo study, model group mice received ethanol intragastrically and normal saline by intramuscular injection. The experimental group received the same dose of alcohol intragastrically and puerarin by intramuscular injection, and the control group received water intragastrically and normal saline by intramuscular injection daily, for 4, 6, 8, and 10 months, respectively. Results: It was found that in the in vitro experimental group, the number of adipocytes, contents of triglycerides and levels of PPARγ mRNA expression were significantly decreased, and alkaline phosphatase activity, contents of osteocalcin and levels of osteocalcin mRNA expression were significantly increased compared with cells in model groups. In the in vivo experimental group, cholesterol, and triglyceride in serum were significantly decreased, and alkaline phosphatase activity was significantly higher, compared with the model group. Fat cell hypertrophy and proliferation, thinner and sparse trabeculae, diminished hematopoiesis, and increased empty osteocyte lacunae in the subchondral region of the femoral head were observed in the model groups. However, no significant changes were seen in femoral heads of the experimental and the control group. Discussion: The results showed that puerarin can inhibit adipogenic differentiation by bone marrow stromal cells both in in vitro in cell culture and in vivo animal experiments. These findings indicate that puerarin can prevent alcohol-induced adipogenesis and osteonecrosis

Objectives. To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H. 2. O. 2. Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10. −6. M – 10. −8. M of estrogen prior to H. 2. O. 2. administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria. Results: H. 2. O. 2. induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H. 2. O. 2. (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H. 2. O. 2. The same experiment was repeated in the presence of bone marrow stromal cells. H. 2. O. 2. increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell. Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM

Angiogenesis is the formation of new blood vessels occurring in an adult through migration and proliferation of endothelial cells, and tubular structures formation. Angiogenesis is modulated by growth factors, cytokines, adhesion molecules, integrins, and enzymes. Angiogenesis plays a role in many physiological processes (i.e. remodeling of ischemic muscle, woumd healing, fracture repair) as well as in pathological process such as rheumathoid arthritis and metastases. In bone, vasculature is essential for cartilage resorption and angiogenesis temporally precedes osteogenesis: the origin of bone is the artery carrying calcium and phosphate ions. Osteogenesis takes place near newly formed vessels, that mediate delivery of osteoprogenitor cells, secrete mitogens for osteoblasts, and transport nutrients and oxygen. Inadequate bone vascularity is associated with decreased bone formation and bone mass. In animals, inhibition of angiogenesis during fracture repair results in the formation of fibrous tissue. A poor blood supply is therefore considered as a risk factor for an impaired bone healing. Angiogenesis is vital in tissue engineering, especially when matrices are colonized by cells with an aerobic metabolism. The scaffold must not only support the growth of the cells making up the organ which should be replaced in vivo (i.e. osteoblasts); it must also support the growth of endothelial cells and develop an effectively functioning vasculature to supply the cells with oxygen. Osteogenesis of tissue engineered materials could be limited by a lack of vascularization, and the bioengineered graft may be potentially resorbed in the same way as a conventional bone graft. In rats, angiogenesis in coralline materials implanted in ectopic muscular sites, was higher when the biomaterial was combined with a vascular pedicle or was coated with bone marrow stromal cells. A combination of both enhanced vascularization and osteogenesis to a greater extent. Endothelial cells release growth factors and cytokines promoting bone deposition: PDGF-AB, TGF-beta 1 and 2, FGF-2, EGF, BMP. However, under inflammatory stimula, endothelial cells release bone resorbing cytokines: IL-6, M-CSF, GM-CSF. Bone marrow stromal cells release angiogenetic proteins such as VEGF, FGF-2, PDGF, TGF, and, after induction with BMP, PlGF. A conversation between bone marrow stromal cells and endothelial cells may therefore be hypothesized. Cultures of bone marrow stromal cells with endothelial cell conditioned medium showed significantly higher phosphatase alkaline activity and osteocalcin production. It was also be hypothesized that stromal cells may acquire immunophenotypic characteristics consistent with endothelial cells. Therefore scaffold requirements are also the ability to favour angiogenesis; endothelial cells growing on the artificial scaffold should mantain a normal phenotype and should not exhibit a pro-inflammatory and bone.resorbing phenotype. Endothelial cell cultures are useful supplementary in vitro tests for the evaluation of scaffolds for bone tissue engineering. Endothelial cell cultures are derived both from animals (usually ox, calf or pig vessels) and from human tissues, mainly the human umbilical vein and the vessels of microcirculation (derma or subcutaneous fat). Endothelial cells in non-human species show different reactions: they have usually a faster replication rate and grow better on the artificial substrata. Endothelial cells from different organs are intrinsically different and exhibit different responses to stimula. if the use of endothelial cells from bone microcirculation should be desirable, they require transfection with viral vectors to be immortalized. To study the response of endothelial cells cultured in vitro on artificial scaffolds, their adhesion, growth, viability and production of metabolites should be evaluated. Adhesion and growth on the materials may be evaluated indirectly by the uptake of Alamar Blue, which measures the amount of oxido-reduction reactions in the cell. A direct evaluation may be obtained by fluorescence microscopy using specific staining for the different cell structures. By studying the expression of adhesins and integrins, the interference of the scaffold with the cell/cell and cell/substrate adhesion should be verified. The release of substances in conditioned medium, as well as the evaluation of specific mRNAs in cells, should be assayed. Among the metabolites released by endothelial cells, the substances promoting bone deposition or favouring resorption, should be investigated. In particular, the release of growth factors may be explored, as they favour cell proliferation and the incorporation of the engineered scaffold within tissues. For the enhancement of bone formation, growth factors may be delivered in different ways: through incorporation on the scaffold, through transfection of bone marrow stromal cells, through platelet gel. Angiogenic growth factors are stored in platelet alpha granules and released during activation. A significant increase in the proliferation of bovine bone endothelial cells was demonstrated after 72 hour incubation with platelet gel in comparison with serum free conditions; the proliferation was similar to the growth induced by the fetal calf serum supplementation (platelet gel: 82.2B18.1x103 cells; serum free: 19.5B11.1x103 cells; fetal calf serum: 72.4B12.4x103 cells). However, the platelet gel inhibited the formation of tubular structures on Matrigel. In conclusion, the development of newly formed vessels on the bone cell engineered scaffold improves the incorporation in the host tissues and the success of the device. The use of exogenous growth factors or of platelet gel favours angiogenesis, besides osteoblast differentiation. The in vitro evaluation of the scaffold should be supplemented by tests on the adhesion, growth and functionality of endothelial cells

Abstract. Objectives. Assess and characterise the suitability of a novel silk reinforced biphasic 3D printed scaffold for osteochondral tissue regeneration. Methods. Biphasic hybrid scaffolds consisted of 3D printed poly(ethylene glycol)-terephthalate-poly(butylene terephthalate)(PEGT/PBT) scaffold frame work (pore size 0.75mm), which has been infilled with a cast and freeze dried porous silk scaffold (5×5×2mm. 3. ), in addition to a seamless silk top layer (1mm). Silk scaffolds alone were used as controls. Both the biphasic and control scaffolds were characterised via uniaxial compression testing (strain rate 0.1mm/min), and the potential biocompatibility of the scaffolds was tested via in vitro culture of seeded bone marrow stromal cells post fabrication. Results. Uniaxial compression testing showed that the biphasic scaffolds (N=4) initially demonstrated similar behaviour on a stress-strain curve to a silk scaffold alone control group (N=6), until a strain of 30% was reached. After 30% strain, load was transitioned from the silk only chondral layer to the 3D printed PEGT/PBT scaffold which resisted further compression and exhibited a significantly greater compressive modulus of 12.6±0.9MPa compared to 0.113±0.01MPa (p<0.001) in the silk scaffold control group. Following 24hours of seeding, no difference was noticed in cell adhesion behaviour under fluorescent microscopy between silk scaffolds and biphasic scaffolds (n=5). Discussion. The use of 3D printing within this novel scaffold provides a solid framework and increases its versatility. The reinforced silk not only provides the secondary Porous structure to the 3D printed scaffold for the bone phase, but also a superficial layer for the cartilage phase. This unique structure has the potential to fill a niche within osteochondral tissue regeneration, especially with the possibility for its use within personalised medicine. Conclusions. These results demonstrate that the novel silk reinforced biphasic 3D printed scaffold is biocompatible and has suitable mechanical properties for osteochondral tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Aims: Collagen implants are used for repair of chondral defects. We investigated the behavior of human chondrocytes of either healthy or osteoarthritic joints and ovine chondrocytes and bone marrow stromal cells seeded in a collagen-GAG copolymer matrix comprising collagen type I, II and III. Methods: Cells were seeded on matrices and cultured for 12 hours, 4 days, 1 week, 2, 3, and 4 weeks. We evaluated morphology and biosynthetic activity of the cells by histological analysis, immunhistochemistry, electron microscopy, biochemical assays for glycosaminoglycans and DNA, and expression of collagens by RT-PCR. Results: From 12 h to 3 weeks the histology showed a increasing number of spherical cells, consistent with chondrocytic morphology except in the osteoarthritic-chondrocyte-seeded scaffolds. GAG analysis showed an increasing amount in all cell-types except osteoarthritic ones. Human chondro-cytes from healthy cartilage increased the amount from 0 μg/mg GAG at 12 hours to 0,9 μg/mg at 2 weeks. Ovine bone marrow stromal cells from 0,5 μg/mg GAG at 12 hours to 2,9 μg/mg at 4 weeks. Conclusions: The collagen trilayer matrix may be of value as a vehicle for chondro-cyte implantation harvested from healthy cartilage. This matrix also supports the expression of chondrocytic proteins in ovine bone marrow stromal cells without use of growth factors. However, chondrocytes from osteoarthritic cartilage revealed low bioactivity and can not be recommended for cell transplantation procedures

Introduction: Ethanol is one of risk factors associated with osteonecrosis, it has been demonstrated that ethanol induces adipogenesis, decreases osteogenesis in bone marrow stroma cells and produces intracellular lipid deposits, resulting in the death of osteocytes. Materials and Methods: In this approach, we isolated human bone marrow stroma cells and triggered for different differentiations. Results: These cells could be induced for osteogenesis, adipogenesis, and chondrogenesis. We also evaluated cell surface markers of isolated human bone marrow stromal cells that were found to express CD29, CD49d, CD62 CD90, CD105/SH2, SH3, CD133, and CD166, but not CD31, CD34, CD45, or CD56. Discussion: We demonstrated that ethanol decreases the expression of osteogenic genes, but increases adipogenic genes expressions. Moreover, we found that ethanol decreases the beta-catenin-dependent canonical Wnt signaling pathway related gene expressions, including Wnt 3a and LRP5 genes. Interestingly, ethanol also diminishes the intra-nuclear translocation of β-catenin in human bone marrow stromal cells. Therefore, these results indicate that ethanol might decrease osteogenic gene expressions through Wnt signaling pathway

Summary. Methotrexate chemotherapy (commonly used in treating cancers and rheumatoid arthritis) creates an inflammatory condition in bone, decreasing osteogenesis, enhancing adipogenesis, increasing osteoclastogenesis, leading to bone loss and marrow adiposity; treatment with fish oil or folinic acid counteracts these negative effects and prevents bone loss. Introduction. Chemotherapy with anti-metabolite methotrexate (MTX) is commonly used in treating cancers and rheumatoid arthritis; however it is known to cause bone loss for which currently there are no adjunct preventative treatments. Methods and Materials. Using a rat model, this study investigated the damaging effects in bones caused by daily MTX injections (0.75mg/kg) for 5 consecutive days (mimicking induction phase treatment for childhood leukaemia) and also the potential protective benefits of omega-3 fatty acid-rich fish oil at different doses (0.25, 0.5 or 0.75 mL/100g BW) in comparison to antidote folinic acid (given i.p at 0.75mg/kg 6 hours post MTX, which is clinically used to reduce MTX toxicities in soft tissues). Results. Histological analysis showed that MTX significantly reduced primary spongiosa bone height and metaphyseal trabecular bone volume. MTX also significantly reduced density of osteoblasts at the secondary spongiosa. Ex vivo differentiation assays with bone marrow stromal cell populations of treated rats revealed a significant reduction in osteogenic differentiation but an increase in adipogenesis. Consistently, RT-PCR gene expression study within the stromal cell population revealed a lower expression of osteogenic transcription factors Runx2 and Osx and bone matrix protein osteocalcin but a significantly upregulated adipogenesis-related genes FABP4 and PPARγ, indicating that MTX chemotherapy induces a switch in the differentiation potential towards adipogenesis at the expense of osteogenesis. MTX increased the density of osteoclasts within the metaphyseal bone as revealed by histological analysis and osteoclast precursor cell pool as shown by ex vivo osteoclastogenesis assay with bone marrow samples. Consistently, mRNA expression of proinflammatory and osteoclastogenic cytokines IL-1, IL-6, TNF-α, and the RANKL/OPG ratio were significantly upregulated by MTX. Supplementary treatment with fish oil (0.5mL/100g BW) or folinic acid significantly preserved metaphyseal trabecular bone volume, osteoblast density, and bone marrow stromal cell osteogenic differentiation and suppressed MTX-induced adipogenesis. These supplements also prevented MTX-induced increased osteoclast density, osteoclastogenesis, and expression of proinflammatory and osteoclastogenic cytokines. Conclusion. These results suggest that MTX chemotherapy creates an inflammatory condition in bone resulting in increased osteoclast formation and decreased osteoblast formation thus leading to bone loss, and that supplementary treatment with fish oil at 0.5mL/100g BW or folinic acid counteract these negative effects, helping to conserve bone formation, suppress bone resorption and bone marrow adiposity, and thus prevent bone loss during MTX chemotherapy

Introduction: Homogenous cell distribution and suffi-cient initial scaffold stability remain key issues for successful tissue engineered osteochondral constructs. The purpose of this study was to investigate the application of initial compression forces during the first 24 hours of cell culture followed by different stress patterns. Methods: Bone marrow stromal cells were harvested from the iliac crest during routine trauma surgery. The cells were expanded in a 2-dimensional culture and then seeded into the biologic hybrid scaffold with a concentration of 1x10E6 cells per ml. Pressure and vacuum forces were applied in a specially developed glass kit. The constructs were exposed to two different protocols of compression combined as oteochondral matrices of CaReS (collagen I) and Tutobone (Ars Arthro, Esslingen, Germany and Tutogen Medical GmbH, Neunkirchen a. Br., Germany). Controls were resected osteochondral fragments from patients with articular fractures and uncompressed constructs. These effects were evaluated using light microscopy after standard staining to identify matrix penetration. Biomechanical tests were conducted, too using a modified biomechanical testing machine. The ‘constrained compression’, maximum load to failure, modulus, and strain energy density were determined. Results: Histology: Penetration and cell distribution was demonstrated homogenous and vital, respectively. Mechanical tests showed a significant enhancement of primary matrix stability. The following stress patterns did not enhance significantly stability over seven days. Discussion: The aim of this project was to investigate the response and cell distrubution of human bone marrow stromal cells seeded on a 3-dimensional biologic hybrid scaffold using compression and vacuum forces. The integration of mechanical stimulation in the tissue engineering process may lead to a progress in the structural and biomechanical properties of these tissues and offers new possibilities in the management of bone injuries and degenerative diseases

Introduction. P-15 (GTPGPQGIAGQRGVV), a fifteen residue synthetic peptide, is a structural analogue of the cell binding domain of Type 1 collagen and creates a biomimetic environment for bone repair when immobilized on anorganic bovine mineral (ABM) scaffolds. ABM-P-15 scaffolds have been shown to enhance bone marrow stromal cell growth and differentiation. This study aimed at evaluating the osteogenic potential of human dental pulp stromal cells (HDPSCs) compared to human bone marrow stromal cells (HBMSCs) in monolayer and on 3D ABM-P-15 scaffolds in vitro and in vivo. Materials and Methods. HDPSCs and HBMSCs were cultured as monolayers in basal or osteogenic media for 3 weeks. Osteogenic differentiation was confirmed using alkaline phosphatase (ALP) staining and ALP specific activity (ALPSA). In addition, the presence and distribution of osteogenic markers including Type 1 collagen, bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OCN) was determined by immunohistochemisty. Gene expression for COL1, RUNX2 and OCN was determined using RT-PCR after 1, 3 and 5 weeks in basal culture. For 3D culture, HDPSCs were seeded on ABM scaffolds ± P-15 (CeraPedics LLC) and cultured in basal media for 6 weeks. Cell viability and growth were visualized by confocal and scanning electron microscopy. Osteogenic differentiation was confirmed by ALP staining and ALPSA. For in vivo studies, HDPSCs were injected and sealed in diffusion chambers containing ABM-P-15 or ABM alone which were then implanted intraperitoneally in nude mice for 8 weeks. The retrieved samples were then processed for histology. Results. In monolayers, HDPSCs showed stronger ALP staining compared to HBMSCs in both culture conditions. Type I collagen, BSP and OPN were detected by immunohistochemistry for both HBMSCs and HDPSCs; however, OCN was not detected. RT-PCR indicated an up regulation of all osteogenic markers in both cell types at weeks 1 and 3. At week 5, there was a marked down regulation of COL1 and RUNX2 in HDPSCS compared to HBMSCs. Confocal microscopy and SEM showed ABM-P-15 promoted HDPSCs bridge formation between the scaffold particles. Histological staining and biochemical analysis confirmed that P-15 enhanced HDPSC ALP activity in vitro and fibrillar collagen formation in vivo compared to ABM alone. Discussion and Conclusion. HDPSCs have higher osteogenic capacity compared to HBMSCs. ABM-P-15 enhanced HDPSC ALPSA and collagen formation, suggesting that a combination of ABM-P15 with HDPSCs could be used as an autologous cell based therapy for bone tissue engineering. Acknowledgement: Supported by a University of Leeds studentships and Cerapedics Inc