Abstract. Objectives. The development of promising therapeutics for cartilage repair/regeneration have been hampered by the inadequacy of existing animal models and lack of suitable translational ex-vivo human tissue models. There is an urgent unmet need for these to assess repair/regenerative (orthobiologic) treatments directly in human tissue. We describe methodology allowing the successful long-term ex-vivo culture of non-degenerate whole human femoral heads that may be used as a model for testing new orthobiologic therapies. Methods. Fifteen fresh, viable human femoral heads were obtained from 15 patients (with ethical permission/consent) undergoing hemiarthroplasty for hip fracture, and cultured aseptically (37°C) for up to 10wks. Culture conditions included static/stirred standard media (Dulbecco's modified Eagle's medium; DMEM) and supplementation with 10% human serum (HS). Chondrocyte viability, density, cell morphology, cell volume, glycosaminoglycan(GAG)/collagen content, surface roughness and cartilage thickness were quantified over time. Results. Chondrocyte viability remained highest (>95%;P<0.01;N(n)=3(12)) under static culture conditons in DMEM+10%HS and was maintained over 10wks. In static DMEM culture without 10%HS, viability remained high for ∼4wks, then decreased rapidly (N(n)=4(16)). Chondrocyte viability declined to <35% over 10wks under all other conditions (N(n)=4(16)). Culturing femoral heads in optimal media (DMEM+10%HS) for 10wks increased the number of chondrocytes producing cytoplasmic processes (P<0.002), but decreased cartilage thickness (P<0.002) and GAG content (P=0.028). Cartilage surface roughness, cellular density, chondrocyte volume and collagen content remained unchanged (P>0.05). Conclusions. The viability of human femoral head articular cartilage could be maintained over 10wks in ex-vivo culture. The model may allow testing of a wide range of orthobiologic therapies directly in human tissue, paving the way for subsequent targeted clinical studies of laboratory-proven strategies with the potential to repair/regenerate articular cartilage. Funder. Chief Scientist's Office, Scotland (Grant TCS/18/01). 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

Geriatric syndromes could lead individuals to exhibit significant mobility and psychological deficits resulting in significant healthcare costs. Thus, identifying strategies to delay aging, or prevent progressive loss of tissue homeostasis could dramatically restore the function and independence of millions of elderly patients and significantly improve quality of life. One of the fundamental properties of aging is the accumulation of senescent cells and senescence associated secretory phenotypes (SASPs) that needs to be treated in wide range of therapeutics including orthobiologics. Senolytic compounds selectively target and kill senescent cells and inhibit anti-apoptotic pathways that are upregulated in senescent cells thereby inducing apoptotic cell death and abrogating systemic SASP factors. We have also shown that blocking fibrosis with Losartan (TGF-β1 blocker) can improve musculoskeletal healing and cartilage repair by reducing the amount of fibrosis. Thus, we hypothesize that administration of anti-fibrotic agents will enhance the beneficial effects of orthobiologics. The safety and efficacy of several senolytic and anti-fibrotic agents to delay age-related dysfunction and improve the function of orthobiologics have been demonstrated in a variety of animal models (in vivo). Overall, our innovative approaches target senescent cells (inflammation) and TGF-β1 (fibrosis) to enhance the clinical efficacy and use of orthobiologics for musculoskeletal repair. We will also discuss ongoing active clinical trials on orthobiologics to aiming at evaluating the safety and efficacy of senolytic agent (Fisetin) and anti-fibrotic agent (Losartan), used independently or in combination, to enhance the beneficial effects of orthobiologics for patients afflicted with musculoskeletal diseases and conditions

Disease modifying approaches are commonly applied in OA patients. An aging society with better life expectancies is increasing in Europe and the globe. Orthobiologics cover intraarticular hyaluronan injections and also cellular therapies. Cellular therapies range from platelet rich plasma (PRP) applications to exosomes. Short term follow-up of limited number of patients revealed favorable results in clinical cellular therapies. Most of these studies evaluated decrease of pain and increase in function. Recent basic science studies focused on the action mechanism of orthobiologic therapies however patient perspective is less studied. Our research team has recently performed a qualitative study on the patient perspective of hyaluronan injection of the knee joint. Findings of that study will be shared and future patient knowledge based options on orthobiologics will be discussed

Due to the presence of megakaryocytes, platelets and clotting factors, bone marrow aspirate (BMA) tends to coagulate. For the first time, starting from our previous studies on mesenchymal vertebral stem cells, it has been hypothesized that coagulated BMA represents a safe and effective autologous biological scaffold for bone regeneration in spinal surgery. The present research involved advanced preclinical in vitro models and the execution of a pilot clinical study. Evaluation of cell morphology, growth kinetics, immunophenotyping, clonogenicity, trilineage-differentiation, growth-factors and HOX and TALE gene expression were analyzed on clotted- and un-clotted human V-BMA. In parallel, a pilot clinical study on ten patients with degenerative spine diseases submitted to instrumented posterior arthrodesis, is ongoing to assess the ability of clotted-V-BMA to improve spinal fusion at 6- and 12-months follow-up. Results demonstrated that clotted-V-BMA have significantly higher growth-factor expression and mesenchymal stem cell (MSCs) viability, homogeneity, clonogenicity, and ability to differentiate towards the osteogenic phenotype than un-clotted-V-BMA. Clotted-V-BMA also highlighted significant reduced expression of PBX1 and of MEIS3 genes negatively involved in osteoblast maturation and differentiation. From December 2020, eight patients have already been enrolled with first promising results that will be finally evaluated in the next two months. The application of V-BMA-clot as carrier of progenitors and cytokines and as natural scaffold with a structural texture represents a point-of-care orthobiologic product to improve spinal fusion. Clinical application seems to be efficacy, and we will confirm and strengthen these data with the final results of the pilot clinical study

Deriving autologous mesenchymal stem cells (MSCs) from adipose tissues without using enzymes requires sophisticated biomedical instruments. Applied pressure on tissues and cells are adjusted manually although centrifugation and filtration systems are frequently used. The number of derived MSCs therefore could differ between instruments. We compared the number of MSCs obtained from four commercially available devices and our newly designed and produced instrument (A2, B3, L3, M2 and T3). Three-hundred mL of adipose tissue was obtained from a female patient undergoing liposuction using the transillumination solution. Obtained tissue was equally distributed to each device and handled according to the producers' guides. After handling, 3 mL stromal vascular fraction (SVF) was obtained from each device. Freshly isolated SVF was characterized using multi-color flow cytometry (Navios Flow Cytometer, Beckman Coulter, USA). Cell surface antigens were chosen according to IFATS and ISCT. CD31-FITC, CD34-PC5,5, CD73-PE, CD90-PB and CD45-A750 (Backman Coulter, USA) fluorochrome-labeled monoclonal antibodies were assessed. Markers were combined with ViaKrome (Beckman Coulter, USA) to determine cell viability. At least 10. 5. cells were acquired from each sample. A software (Navios EX, Beckman Coulter, USA) was used to create dot plots and to calculate the cell composition percentages. The data was analyzed in the Kaluza 2.1 software package (Beckman Coulter, USA). Graphs were prepared in GraphPad Prism. CD105 PC7/CD31 FITC cell percentages were 23,9%, 13,5%, 24,6%, 11,4% and 28,8% for the A2, B3, L3, M2 and T3 devices, respectively. We conclude that the isolated MSC percentage ranged from 11,4% to 28,8% between devices. The number of MSCs in SVF are key determinants of success in orthobiological treatments. Developing a device should focus on increasing the number of MSCs in the SVF while preserving its metabolic activity. Acknowledgments: Scientific and Technological Research Council of Türkiye (TÜBİTAK)- Technology and Innovation Funding Program Directorate (TEYDEB) funded this project (#321893). Servet Kürümoğlu and Bariscan Önder of Disposet Ltd., Ankara, Türkiye (. www.disposet.com. ) contributed to the industrial design and research studies. Ali Tuncel and Feza Korkusuz are members of the Turkish Academy of Sciences (TÜBA). Nilsu Baysal was funded by the STAR Program of TÜBITAK Grant # 3210893

Summary. The BMP-2 content and bone forming potential of 2 leading allograft products (OsteoAMP® and Osteocel® Plus) was tested across 3 commercially available lots. Surprisingly, there was no BMP-2 content associated with the cells contained within Osteocel® Plus. OsteoAMP® contained greater than 1000 times the overall BMP-2 content than Osteocel® Plus. Correspondingly, Osteocel® Plus did not form new bone at any timepoint in the 12 week in vivo study while OsteoAMP® had increasing new bone formation at each sequential timepoint. Interestingly, the highest cellularity of Osteocel® Plus was just prior to implant at t. 0. , decreasing at each timepoint, decreasing further at the terminal endpoint of the study at 12 weeks (82% of cells had died or migrated). Conversely, the cellularity of OsteoAMP® increased at each timepoint. Introduction. Implants containing living cells are often characterised by the orthobiologics industry as ‘osteogenic’. The positive function and ultimate fate of these cells has been assumed with little to no proof of efficacy. In this study we compare the bone forming ability of the market leading stem cell product claiming osteoinductivity as well as osteogenicity, Osteocel® Plus, against the market leading allograft derived growth factor product, OsteoAMP® which claims osteoinductivity but contains no viable cells. The goal of the study is to determine if a cellular product will form new bone or produce a false positive when evaluated histomorphometrically using an osteoinductive control over time in vivo. Additionally, the osteoinductive potential from each product will be quantified by in vitro by measurement of BMP-2 content via ELISA. Methods. Three different lots of each OsteoAMP® and Osteocel® Plus were implanted by an independent lab into muscle pouches of athymic rats. Implants were assessed for bone formation using H&E histology at time of implant, 1 day, 2w, 4w, 6w, 8w, and 12w. Histomorphometric measurements were done using Image J (NIH) using four sections per implant per timepoint (n=12). Each lot of both products was also digested in water, hydrochloric acid, and guanidine-hydrochloride (Gua-HCl) to facilitate BMP-2 content quantification via ELISA (R&D Systems). The extracts were intended to independently measure cellular, mineral, and collagen phase content of BMP-2. All extracts were analyzed by ELISA independently to characterise the source of osteoinductivity and the resulting release profile in vivo. Results. With each subsequent timepoint, all lots of Osteocel® Plus exhibited increasing osteonecrosis with increasing time. By the terminal 12 week timepoint used in this study, 82% of living bone that was implanted for Osteocel® Plus had died or migrated away from the implant site. In contrast, new bone formation and cellularity increased with OsteoAMP® at each timepoint, still increasing at the terminal 12 week endpoint of this study. The BMP-2 results support the bone formation in vivo. Neither of the 3 Osteocel® Plus lots registered any BMP-2 from the cells on ELISA, nor was there any free BMP-2 that leached out of the product. The mineral phase of Osteocel® Plus exhibited traces of BMP-2 for only one of the 3 donors with an average of 0.04 ng/g for all three donors. The protein phase of Osteocel® Plus had the highest concentration of BMP-2 at 0.63 ng/g BMP-2. For all three lots, OsteoAMP® showed no leaching of BMP-2 after exposure to water and a BMP-2 content in the mineral phase of 50.85 ng/g. The collagen phase of OsteoAMP® had the highest concentration of BMP-2 in all groups tested at 915.54 ng/g. Discussion. The results in this study would suggest that orthobiologic implants that contain living cells and claim osteogenic properties do not maintain viable cells over time and did not form bone. Similarly, the results would indicate the living cells do not contribute to osteoinductivity. In contrast, the higher BMP-2 content and osteoinductive property exhibited by OsteoAMP® was a stronger producer of new bone

Surgical repair of rotator cuff tears have high failure rates (20–70%), often due to a lack of biological healing. Augmenting repairs with extracellular matrix-based scaffolds is a common option for surgeons, although to date, no commercially available product has proven to be effective. In this study, a novel collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. The collagen scaffold was assessed in vitro for cytocompatability and retention of tenocyte phenotype using alamarBLUE assays, confocal imaging and real-time PCR. Immunogenicity was assessed in vitro by the activation of pre-macrophage cells. In vivo, using a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 46 animals. Overlay augmentation with the collagen scaffold was compared to unaugmented repairs. At 6- and 12-weeks post-op the repairs were tested biomechanically to evaluate repair strength, and histologically for quality of healing. The collagen scaffold supported human tenocyte growth in vitro, with cells appearing morphologically tenocytic and expressing higher tendon gene markers compared to plastic controls. No immunogenic responses were provoked compared to suture material control. In vivo, augmentation with the scaffold improved the histological scores at 12 weeks (8.37/15 vs. 6.43/15, p=0.0317). However, no significant difference was detected on mechanical testing. While the collagen scaffold improved the quality of healing of the tendon, a meaningful increase in biomechanical strength was not achieved. This is likely due to its inability to affect the bone-tendon junction. Future materials/orthobiologics must target both the repaired tendon and the regenerating bone-tendon junction