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Introduction: Osteochondral (OC) disorders like osteoarthritis (OA) and rheumatoid arthritis (RA) damage the joint's cartilage and subchondral bone. Their treatment remains a significant challenge for both researchers and orthopedics. In vitro models of OC tissue have become an essential tool to help investigate pathogenesis, develop drug screening, and test potential therapeutic approaches. This study aims to create a bio-printed OC construct recapitulating the bone and cartilage compartment as drugs testing platforms. Methodology: Two different hydrogels including a blend composed of gelatin methacrylate (GelMA) with nanosized hydroxyapatite (nHA) and tyramine-modified hyaluronic acid (THA) were selected for the bioprinting of bone and cartilage tissue mimics. The composition of GelMA hydrogel (10% w/v) with different concentrations of nHA (1-10% w/v) and THA with concentrations of 2.5-5% w/v were characterized by rheology and their cytotoxicity was assessed via live-dead assay. Later, the pre-differentiated osteoblast and endothelial cells were encapsulated into GelMA-nHA and micropellet chondrocytes into THA hydrogels for bioprinting osteochondral construct. After 2 weeks of culturing, the successful generation of OC tissue was confirmed by real-time RT-PCR and histology. Results: The storage modulus (G') of all GelMA/nHA hydrogels was significantly higher than GelMA, however, there was no significant difference in G' values for the GelMA/nHA as a function of added nHA. Due to the know temperature sensitivity of GelMA, a rheological temperature sweep and series of printing tests were performed to establish a suitable printing temperature, which was confirmed to be 20°C, independent of the addition of nHA. Calcein-AM (Ca-AM) and Ethidium Homodimer-1 (EthD-1) staining for GelMA (10% w/v) with three concentrations of nHA (1, 3, and 5% w/v) at 2, 24, 72, and 168 h after printing showed the percentage of living cells after 72h in GelMA containing 3 and 5% (w/v) nHA was less than 50%, while in GelMA with 1% (w/v) nHA it remained high (>95%) even after 168h. Therefore, this formulation was chosen for the subsequent generation of bone tissue mimic. Shear flow curves of THA hydrogels showed an increase in viscosity as a function of THA concentration. The damping factor, which is a ratio between the loss modulus G'' and the storage modulus G', has been shown to be directly related to the extrudability. The calculated damping factors for each concentration of THA (%w/v) (THA 2.5%= 0.4947 ± 0.038, THA3.5%= 0.5935 ± 0.012, and THA5%= 0.7391 ± 0.039), indicated that THA3.5%(w/v) was in the printable range. Cell viability assays for THA hydrogels showed a high percentage of living cells for THA 3.5% (w/v) compared to THA 5% (w/v) after 168 h. Based on cell viability assay, viscosity, and printability, a 3.5%(w/v) concentration of THA was selected for generating cartilage tissue mimic part. Conclusion: We developed GelMA-nHA and THA hydrogels for bone and cartilage parts respectively. We also optimized printing parameters based on printability and shape fidelity and cell density according to cell viability for bioprinting OC constructs.

Our recent study detected the expression of a tissue renin���angiotensin system (tRAS) in human intervertebral discs (IVDs). The present study sought to investigate the impact of the angiotensin II receptor type 1 (AGTR1) antagonist losartan on human nucleus pulposus (NP) cell inflammation and degeneration induced by tumor necrosis factor-�� (TNF-��). Human NP cells (4 donors; Pfirrmann grade 2���3; 30���37-years���old; male) were isolated and expanded. TNF-�� (10 ng/mL) was used to induce inflammation and degeneration. We examined the impact of losartan supplementation and measured gene expression of tRAS, anabolic, catabolic, and inflammatory markers in NP cells after 24 and 72 h of exposure. T0070907, a PPAR gamma antagonist, was applied to examine the regulatory pathway of losartan. Losartan (1 mM) significantly impaired the TNF-��-induced increase of pro-inflammatory (nitric oxide and TNF-��), catabolic (matrix metalloproteinases), and tRAS (AGTR1a and angiotensin-converting enzyme) markers. Further, losartan maintained the NP cell phenotype by upregulating aggrecan and downregulating collagen type I expression. In summary, losartan showed anti-inflammatory, anti-catabolic, and positive phenotype-modulating effects on human NP cells. These results indicate that tRAS signaling plays an important role in IVD degeneration, and tRAS modulation with losartan could represent a novel therapeutic approach.

Additional file 4 : Supplementary Table 5 Concentrations of cytokines and chemokines in pooled conditioned media from healthy, traumatic and degenerative intervertebral disc, measured by immunoassay technique (mean+/-sd of technical replicates; pg/mL).

Supplemental Material, sj-pdf-1-gsj-10.1177_21925682211058158 for Research Practices and Needs Among Spine Surgeons Worldwide by Niccole Germscheid, Jason P. Y. Cheung, Marko H. Neva, F. Cumhur ��ner, Brian K. Kwon, Marcelo Valacco, Waleed Awwad, Daniel M. Sciubba, Stephen J. Lewis, Laurence D. Rhines, Sangwook T. Yoon, Mauro Alini, Sibylle Grad, Charles Fisher and Dino Samartzis in Global Spine Journal

Supplemental Material, sj-pdf-2-gsj-10.1177_21925682211058158 for Research Practices and Needs Among Spine Surgeons Worldwide by Niccole Germscheid, Jason P. Y. Cheung, Marko H. Neva, F. Cumhur ��ner, Brian K. Kwon, Marcelo Valacco, Waleed Awwad, Daniel M. Sciubba, Stephen J. Lewis, Laurence D. Rhines, Sangwook T. Yoon, Mauro Alini, Sibylle Grad, Charles Fisher and Dino Samartzis in Global Spine Journal

Additional file 3 : Supplementary Table 1. MSC secretome following healthy CM stimulation. Supplementary Table 2. MSC secretome following traumatic CM stimulation. Supplementary Table 3. MSC secretome following degenerative CM stimulation. Supplementary Table 4. MSC secretome following IL-1β stimulation.

Supplemental Material, sj-pdf-2-gsj-10.1177_21925682211058158 for Research Practices and Needs Among Spine Surgeons Worldwide by Niccole Germscheid, Jason P. Y. Cheung, Marko H. Neva, F. Cumhur ��ner, Brian K. Kwon, Marcelo Valacco, Waleed Awwad, Daniel M. Sciubba, Stephen J. Lewis, Laurence D. Rhines, Sangwook T. Yoon, Mauro Alini, Sibylle Grad, Charles Fisher and Dino Samartzis in Global Spine Journal

Background Mesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes. Methods This study aimed to investigate the proteome released by bone marrow-derived MSCs following exposure to a healthy, traumatic, or degenerative human IVD environment by mass spectroscopy and quantitative immunoassay analyses. Exposure of MSCs to the proinflammatory stimulus interleukin 1β (IL-1β) was used as control. Results Compared to MSC baseline secretome, there were 224 significantly up- or downregulated proteins following healthy, 179 following traumatic, 223 following degenerative IVD, and 160 proteins following IL-1β stimulus. Stimulation of MSCs with IVD conditioned media induced a more complex MSC secretome, involving more biological processes, compared to stimulation with IL-1β. The MSC response to stimulation with IVD conditioned medium was dependent on their pathological status. Conclusions The MSC secretome seemed to match the primary need of the IVD: homeostasis maintenance in the case of healthy IVDs, versus immunomodulation, adjustment of ECM synthesis and degradation disbalance, and ECM (re) organization in the case of traumatic and degenerative IVDs. These findings highlight the importance of cell preconditioning in the development of tailored secretome therapies., Stem Cell Research & Therapy, 12 (1)

Additional file 1: Supplemental Table S1. List of primers by using SyberGreen for qRT- PCR. Supplemental Table S2. Primer and probe sequences for qRT- PCR. Supplemental Table S3. List of assays on demand used for qRT-PCR.

Additional file 2: S-Fig. 1- Immunohistochemical staining against to Coll II and Coll X. S-Fig. 2. Flow cytometric analysis of CD markers in human BM-MSCs at passage 3. The majority of the cells expressed the typical CD markers related to MSC (CD 44 and CD 90, CD73, CD105). Other antigens were also expressed in a minority of the cells. S-Fig. 3. Differentiation potential of the isolated cells from human Bone Marrow. A) Sections prepared from micromass culture for chondrogenesis stained purple following toluidine blue staining, B) Osteogenic culture stained red following alizarin red staining. C) Adipogenic culture stained red following oil red staining. Culture conditions and staining methods were as previously described (Meury at al., Cell Biochem. 2006 Jul 1;98(4):992–1006).

Supplemental material, DS_10.1177_0363546519887909 for Articular Joint-Simulating Mechanical Load Activates Endogenous TGF-β in a Highly Cellularized Bioadhesive Hydrogel for Cartilage Repair by Peter Behrendt, Yann Ladner, Martin James Stoddart, Sebastian Lippross, Mauro Alini, David Eglin and Angela Rita Armiento in The American Journal of Sports Medicine

Angiogenesis is a key factor in early stages of wound healing and is crucial for the repair of vascularized tissues such as the bone. However, supporting timely revascularization of the defect site still presents a clinical challenge. Tissue engineering approaches delivering endothelial cells or prevascularized constructs may overcome this problem. In the current study, we investigated platelet-rich plasma (PRP) gels as autologous, injectable cell delivery systems for prevascularized constructs. PRP was produced from human thrombocyte concentrates. GFP-expressing human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in PRP gels in different proportions. The formation of cellular networks was assessed over 14 days by time-lapse microscopy, gene expression analysis, and immunohistology. PRP gels presented a favorable environment for the formation of a three-dimensional (3D) cellular network. The formation of these networks was apparent as early as 3 days after seeding. Networks increased in complexity and branching over time but were only stable in HUVEC-MSC cocultures. The high cell viability together with the 3D capillary-like networks observed at early time points suggests that PRP can be used as an autologous and proangiogenic cell delivery system for the repair of vascularized tissues such as the bone.

Articular cartilage displays very little self-healing capabilities, generating a major clinical need. Here, we introduce a thermoresponsive hyaluronan hydrogel for cartilage repair obtained by covalently grafting poly(N-isopropylacrylamide) to hyaluronan, to give a brush co-polymer HpN. The gel is fluid at room temperature and becomes gel at body temperature. In this pilot study HpN safety and repair response were evaluated in an osteochondral defect model in rabbit. Follow-up was of 1 week and 12 weeks and the empty defect served as a control, for a total of four experimental groups. At 12 weeks the defect sites were evaluated macroscopically and histologically. Local lymph nodes, spleen, liver, and kidneys were analyzed for histopathological evaluation. HpN could be easily injected and remained into the defect throughout the study. The macroscopic score was statistically superior for HpN versus empty. Histological score gave opposite trend but not statistically significant. A slight tissue reaction was observed around HpN, however, vascularization and subchondral bone formation were not impeded. An upper proteoglycans rich fibro-cartilaginous tissue with fairly good continuity and lateral integration into the existing articular cartilage was observed in all cases. No signs of local or systemic acute or subacute toxicity were observed. In conclusion, HpN is easily injectable, remains into an osteochondral defect within a moving synovial joint, is biocompatible and does not interfere with the intrinsic healing response of osteochondral defects in a rabbit model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1469-1478, 2016.

Despite its remarkable ability to resist mechanical loading, articular cartilage is not capable of mounting a useful reparative reaction in response to damage caused by trauma or disease. As a result numerous surgical and medical approaches have been developed to aid the healing of articular cartilage. Despite the success of surgical techniques such as microfracture, recently attentions have been turned to cell based therapies such as autologous chondrocyte implantation (ACI). ACI has produced encouraging results, however better results may be achievable through an evolution of this surgical approach. Since the first generation of ACI techniques changes have been made in the technique e.g. the introduction of collagen membranes instead of periosteal flaps, and more recently the use of collagen scaffolds for cellular delivery. The procedure has also moved on from being performed as an open operation and can now be performed arthroscopically. Despite these advances the procedure still uses chondrocytes harvested from the joint being repaired. These cells are vulnerable to dedifferentiation during the required in vitro expansion, and as a result may not be capable of producing repair tissue once implanted back into the joint. Mesenchymal stem cells (MSCs) may provide a dedifferentiation resistant alternative to chondrocytes. MSCs would also allow for the use of one arthroscopic operation on the affected joint, as opposed to the two operations that are currently required for ACI.

Tissue engineering repair of annulus fibrosus (AF) defects has the potential to prevent disability and pain from intervertebral disc (IVD) herniation and its progression to degeneration. Clinical translation of AF repair methods requires assessment in long-term large animal models. An ovine AF injury model was developed using cervical spinal levels and a biopsy-type AF defect to assess composite tissue engineering repair in 1-month and 12-month studies. The repair used a fibrin hydrogel crosslinked with genipin (FibGen) to seal defects, poly(trimethylene carbonate) (PTMC) scaffolds to replace lost AF tissue, and polyurethane membranes to prevent herniation. In the 1-month study, PTMC scaffolds sealed with FibGen herniated with polyurethane membranes. When applied alone, FibGen integrated with the surrounding AF tissue without herniation, showing promise for long-term studies. The 12-month long-term study used only FibGen which showed fibrous healing, biomaterial resorption and no obvious hydrogel-related complications. However, the 2 mm biopsy punch injury condition also exhibited fibrotic healing at 12 months. Both untreated and FibGen treated groups showed equivalency with no detectable differences in histological grades of proteoglycans, cellular morphology, IVD structure and blood vessel formation, biomechanical properties including torque range and axial range of motion, Pfirrmann grade, IVD height, and quantitative scores of vertebral body changes from clinical computed tomography. The biopsy-type injury caused endplate defects with a high prevalence of osteophytes in all groups and no nucleus herniation, indicating that the biopsy-type injury requires further refinement, such as reduction to a slit-type defect that could penetrate the full depth of the AF without damaging the endplate. Results demonstrate translational feasibility of FibGen for AF repair to seal AF defects, although future study with a more refined injury model is required to validate the efficacy of FibGen before translation.