Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Pre-treatment with simvastatin significantly reduced cobalt-mediated IL-8 protein secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells (p-value<0.0001). Work will be undertaken to determine changes in gene expression, the role of TLR4 in these responses and the effect of simvastatin on additional inflammatory markers. Conclusions. Simvastatin significantly reduces cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving aseptic loosening and pseudotumour formation

We investigated the circulating levels of the main cytokines involved in bone resorption (IL-1β, IL-6, TNF-α), prostaglandins (PGE. 2. ) and metalloproteases (MMP-1), as possible early markers of osteolysis, in the serum of eight patients with periprosthetic osteolysis and ten patients without osteolysis. All had received a cementless hip prosthesis (ABG-1). We also assessed the serum levels of IL-11 and TGF-β anti-inflammatory cytokines exerting protective effect on bone resorption. The mean serum levels of IL-1β, IL-6, TNF-α, TGF-β, MMP-1, and PGE. 2. in patients with periprosthetic osteolysis did not differ significantly from those of patients without osteolysis or from those of normal controls. IL-11 serum levels were not detectable at all in any of the patients, while they were detected within normal reference values in the control subjects (significant inverse correlation). We believe that circulating cytokines cannot be regarded as markers of osteolysis, a condition characterised by a local inflammation without systemic signs of inflammation. On the contrary, the undetectable levels of IL-11 in implanted patients could provide evidence for a lack of balance between pro- and anti-inflammatory cytokines in these patients

BACKGROUND. Although osteoarthritis (OA) is not an inflammatory arthritis, a characteristic feature of OA is increased production of pro-inflammatory cytokines, such as interleukin 1beta (IL-1b), by articular chondrocytes. In fact, the degree of articular inflammation is often associated with disease progression; indicating that this process probably contributes to articular damage. Suppressor of cytokine signalling (SOCS) proteins are, as the name suggests, inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain-1 with similar structure to the other SOCS proteins) have been identified, of which, SOCS1-3 and CIS-1 are the best characterised. Reduced expression of SOCS proteins would be predicted to result in increased cytokine responsiveness and thereby could contribute to OA pathology. OBJECTIVES. 1) To compare the expression of SOCS1-3 and CIS-1 in normal and OA human articular chondrocytes and 2) to analyze the effects of IL-1b on SOCS1-3 and CIS-1 mRNA expression. METHODS. Femoral heads were obtained after joint replacement surgery due to OA or following a fracture of the neck of femur (#NOF). The latter patients typically suffer from osteoporosis and their cartilage is widely used as a suitable non-OA control. Chondrocytes from the surface layer of OA or the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total RNA was extracted using the Qiagen AllPrep RNA mini kit. Primers were designed for SOCS1-3 and CIS-1 and relative gene expression was determined by real-time RT-PCR (Sybr-green method), normalized to GAPDH. Alternatively, non-OA chondrocytes were isolated from #NOF cartilage and cultured in DMEM/F12/5% FCS, with or without IL-1b plus oncostatin M (OSM, both 2.5 ng/ml) for 3-5 weeks until confluent. Total RNA was extracted as above and the effect of IL-1b on gene expression was determined by qPCR. RESULTS. Expression of SOCS1 and SOCS3 was similar in OA and #NOF chondrocytes. However the expression of SOCS2 and CIS-1 was reduced 13-fold in OA samples compared to #NOF samples (n=5, each group). Expression of one OA patient was set to =1 to determine relative expression. In #NOF chondrocytes, relative expression of SOCS2 was 24.5±20.4 versus 2.5±1.9 in OA chondrocytes and for CIS-1 the values were 26.5±19.5 versus 2.5±1.8 (p<0.01). The in vitro experiments showed that treatment of control #NOF chondrocytes with IL-1b+OSM mimicked the situation in OA: Expression of SOCS2 in cytokine-treated cultures was only 15±10% of that in control cultures and CIS-1 expression was reduced to 16±13% (P<0.01). CONCLUSIONS. To the authors' knowledge this is the first demonstration of reduced expression of SOCS2 and CIS-1, but not SOCS1 or SOCS3, in osteoarthritis. As the SOCS proteins are attenuators of cytokine-mediated processes, reduced expression would be expected to accelerate any inflammatory processes. The fact that IL-1b itself reduces the expression of its suppressors of signalling suggests a potentially damaging positive feedback mechanism that could play a role in OA pathology

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Frozen shoulder is a chronic fibrosing condition of the capsule of the joint. The predominant cells involved are fibroblasts and myofibroblasts which lay down a dense matrix of type-I and type-III collagen within the capsule. This subsequently contracts leading to the typical features of pain and stiffness. Cytokines and growth factors regulate the growth and function of the fibroblasts of connective tissue and remodelling of the matrix is controlled by the matrix metalloproteinases (MMPs) and their inhibitors. Our aim was to determine whether there was an abnormal expression or secretion of cytokines, growth factors and MMPs in tissue samples from 14 patients with frozen shoulder using the reverse transcription/polymerase chain reaction (RT/PCR) technique and to compare the findings with those in tissue from four normal control shoulders and from five patients with Dupuytren’s contracture. Tissue from frozen shoulders demonstrated the presence of mRNA for a large number of cytokines and growth factors although the frequency was only slightly higher than in the control tissue. The frequency for a positive signal for the proinflammatory cytokines Il-1β and TNF-α and TNF-β, was not as great as in the Dupuytren’s tissue. The presence of mRNA for fibrogenic growth factors was, however, more similar to that obtained in the control and Dupuytren’s tissue. This correlated with the histological findings which in most specimens showed a dense fibrous tissue response with few cells other than mature fibroblasts and with very little evidence of any active inflammatory cell process. Positive expressions of the mRNA for the MMPs were also increased, together with their natural inhibitor TIMP. The notable exception compared with control and Dupuytren’s tissue was the absence of MMP-14, which is known to be a membrane-type MMP required for the activation of MMP-2 (gelatinase A). Understanding the control mechanisms which play a part in the pathogenesis of frozen shoulder may lead to the development of new regimes of treatment for this common, protracted and painful chronic fibrosing condition

Summary. Cytokines produced within the degenerate disc induce expression of neurotrophic factors and pain related peptides which could be important in nerve ingrowth and pain sensitisation leading to low back pain. The intervertebral disc (IVD) is considered the largest aneural and avascular structure within the human body, yet during degeneration vascularisation of the IVD is seen to be accompanied by nociceptive nerves. Low back pain is a highly debilitating condition affecting around 80% of the population, 40% of which are attributed to IVD degeneration. Discogenic pain was largely thought to be a result of irritation and compression of the nerve root, yet recent data suggests that pain may be attributed to the sensitisation of sensory nerves by the synthesis of pain related peptides, calcitonin gene related peptide (CGRP) and substance P. It is known that cytokines and chemokines produced by nucleus pulposus cells elicit various effects including the production of matrix degrading enzymes, and decreased matrix molecules. Here, we investigate the hypothesis that cytokines regulate both neurotrophic factor and pain related peptide synthesis within nucleus pulposus and nerve cells which may elicit algesic effects. Real-Time PCR was performed to investigate gene expression of the neurotrophic factors NGF, BDNF, NT3 and their receptors Trk A, B and C along with Substance P and CGRP on directly extracted RNA from human NP cells and NP cells cultured in alginate for 2 weeks prior to treatment for 48hours with IL-1, IL-6 or TNFα at 0–100ng/mL. Similarly SH-SY5Y neuroblastoma cells were differentiated in retinoic acid for 7 days prior to stimulation with IL-1, IL-6 or TNFα at 0ng/mL and 10ng/mL for 48hours. Immunohistochemistry was used to localise neurotrophic factor receptors Trk A, B and C in both degenerate discs and neuronal cells. NGF expression was present in normal and degenerate disc samples, however only degenerate discs expressed the high affinity receptor TrkA. Similarly Trk B was present in 22% of normal samples increasing to 100% expression within degenerate disc samples. All cytokines increased expression of NGF in NP cells (P≤0.05). TNFα also increased BDNF significantly, whereas no significant affects were seen in NT3 expression in NP cells. Trk B expression was significantly increased by IL-1 and TNFα treatment of NP cells. Conversely Trk C was down regulated by IL-6. Substance P was significantly increased by IL-1 and TNFα treatments whilst IL-6 and TNFα increased CGRP expression in NP cells. In SH-SY5Y cells, IL-1 significantly increased BDNF whilst IL-6 and TNFα failed to induce significant differences in neurotrophic factors. All cytokines increased Trk expression in the nerve cell line; however this failed to reach significance. Immunohistochemistry confirmed the presence of Trk receptors within the neuronal cell line. Here we have demonstrated that a number of cytokines known to be up regulated during disc degeneration and disc prolapse, induce expression of various neurotrophic factors, their receptors and pain related peptides within human NP cells, as well as SH-SY5Y cells. This data suggests that the presence and production of cytokines within the degenerate disc may be responsible for nerve ingrowth and sensitisation of nerves which may result in discogenic pain

Particulate prosthetic materials are often studied by adding them to monocytic cells in vitro and measuring the release of cytokines as an indicator of their inflammatory potential. Endotoxin is known to be a contaminant of particle preparations and also stimulates the release of cytokines. It is usual to use a proprietary endotoxin test to avoid erroneous results. Four different formulations of cement were found to be free from endotoxin using standard, gelclot tests but stimulated different levels of release of cytokines from macrophages. These differences were explained when a more sensitive, kinetic endotoxin assay showed that release correlated with minor contamination with endotoxin. In a repeat experiment using cement particles with low or undetectable levels of endotoxin by kinetic assay, differences in the ability of the formulations to stimulate the release of cytokines were not seen. Endotoxin is adsorbed on to the surface of particles and it is this combination which stimulates increased release of cytokines. In both the above methods for determination of endotoxin, the water in which the particles had been soaked was examined rather than the particles directly. Further investigations showed that a kinetic assay directly on a particle suspension is the most sensitive method to measure contamination with endotoxin

The tissues surrounding 65 cemented and 36 cementless total joint replacements undergoing revision were characterised for cell types by immunohistochemistry and for cytokine expression by in situ hybridisation. We identified three distinct groups of revised implants: loose implants with ballooning radiological osteolysis, loose implants without osteolysis, and well-fixed implants. In the cemented series, osteolysis was associated with increased numbers of macrophages (p = 0.0006), T-lymphocyte subgroups (p = 0.03) and IL-1 (p = 0.02) and IL-6 (p = 0.0001) expression, and in the cementless series with increased numbers of T-lymphocyte subgroups (p = 0.005) and increased TNFα expression (p = 0.04). For cemented implants, the histological, histochemical and cytokine profiles of the interface correlated with the clinical and radiological grade of loosening and osteolysis. Our findings suggest that there are different biological mechanisms of loosening and osteolysis for cemented and cementless implants. T-lymphocyte modulation of macrophage function may be an important interaction at prosthetic interfaces

Background. Adverse reactions to metal debris are implicated in the failure of metal-on-metal hip arthroplasty. The peri-implant tissues are often infiltrated by leukocytes which may cause observed immunological effects, including soft tissue necrosis and osteolysis. Cobalt ions from orthopaedic implants aberrantly activate the innate immune receptor human toll-like receptor-4 (TLR4), leading to inflammatory cytokine release including interleukin-8 (IL-8). IL-8 has been shown to increase expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These factors are essential for leukocyte adhesion to endothelium, which is required for leukocyte migration into tissues. This study investigates cobalt's effect on gene and protein changes in IL-8, ICAM-1 and VCAM-1 to determine their potential role in immune cell infiltration of peri-implant tissues. Methods. TLR4-expressing human dermal microvascular endothelial cells (HMEC-1) were treated with a range of clinically relevant cobalt ion concentrations. IL-8 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Stimulation with cobalt ions significantly increases IL-8 secretion (n=3) in HMEC-1 cells. This is a TLR4-specific effect as a small molecule TLR4 antagonist inhibited cobalt-induced IL-8 secretion. Following cobalt treatment (0.75mM cobalt chloride) there is a 12-fold increase in ICAM-1 (p-value=0.0004) and a 6-fold increase in VCAM-1 (p-value<0.0001) gene expression. Work will be undertaken to determine the role of TLR4 in these responses. Conclusion. Cobalt increases IL-8 secretion and adhesion molecule gene expression in HMEC-1 cells. This in vitro finding demonstrates the potential for cobalt ions to increase leukocyte adhesion to the endothelial surface. This may contribute to leukocyte infiltration of peri-implant tissues in metal-on-metal hip arthroplasty failure

Our aim was to determine if the serum levels of bone-resorbing cytokines (IL-1β, TNF-α, IL-6, GM-CSF) are altered in patients with aseptic loosening of a total hip prosthesis, and if such levels are influenced by the type of implant. We determined cytokine levels in sera from 35 patients before revision for failed total hip arthroplasty and compared them with those in 25 healthy donors. We also assessed the soluble receptor of interleukin-2 (sIL-2r) in serum as an indication of a specific immune reaction against the implant. Our findings showed that the sIL-2r and TNF-α serum level did not change. The IL-6 level was not significantly altered, but was higher in patients with TiAlV prostheses than in those with a CrCoMo implant and in patients with cemented prostheses. The IL-1β level was found to be higher in those with a TiAlV cemented prosthesis than in the control group (p = 0.0001) and other groups of patients (p = 0.003 v uncemented TiAlV, p = 0.01 v cemented CrCoMo, p = 0.001 v uncemented CrCoMo). The GM-CSF level significantly increased in patients compared with healthy subjects (p = 0.008), and it was higher in those with cemented than with uncemented implants (p = 0.01). Only patients with cementless CrCoMo prostheses had levels of GM-CSF similar to those of the control group. The highest GM-CSF concentrations were observed in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) in the last months before revision (p = 0.04). In addition, when massive osteolysis was observed, the level of GM-CSF tended to decrease to that of the control group

Proponents of the biological theory of aseptic loosening have in recent years tended to concentrate on the production and distribution of particulate ultra-high-molecular-weight polyethylene (UHMWPE) debris around the potential joint space. However, mechanical loading of cemented implants with the differing elastic moduli of metal stems, polymethylmethacrylate (PMMA) cement and bone can result in relative micromotion, implying the potential for production of metal and PMMA particles from the stem-cement interface by fretting wear. In order to investigate the production and biological reactivity of debris from this interface, PMMA and metal particulate debris was produced by sliding wear of PMMA pins containing barium sulphate and zirconium dioxide against a Vaquasheened stainless steel counterface. This debris was characterised by SEM, energy-dispersive analysis by X-ray (EDAX) and image analysis, then added to cell cultures of a human monocytic cell line, U937, and stimulation of pro-osteolytic cytokines measured by ELISA. Large quantities of PMMA cement debris were generated by the sliding wear of PMMA pins against Vaquasheened stainless steel plates in the method developed for this study. Both cements stimulated the release of pro-osteolytic TNFα from the U937 monocytic cell line, in a dose-dependent fashion. There was a trend towards greater TNFα release with Palacos cement than CMW cement at the same dose. Palacos particles also caused significant release of IL-6, another pro-osteolytic cytokine, while CMW did not. The particulate cement debris produced did not stimulate the release of GM-CSF or IL1β from the U937 cells. These results may explain the cytokine pathway responsible for bone resorption caused by particulate PMMA debris. Radio-opaque additives are of value in surgical practice and clinical studies to quantify the relevance of these in vitro findings are required before the use of cement containing radio-opacifier is constrained

We analysed synovial fluid from 88 hips, 38 with osteoarthritis and 12 with well-functioning and 38 with loose hip prostheses. The levels of TNF-α, IL-1ß (71 hips) and IL-6 (45 hips) were measured using the ELISA technique. Joints with well-functioning or loose prostheses had significantly increased levels of TNF-α compared with those with osteoarthritis. Hips with aseptic loosening also had higher levels of IL-1ß but not of IL-6 compared with those without an implant. The levels of TNF-α and IL-1ß did not differ between hips with stable and loose prostheses. Higher levels of TNF-α were found in hips with bone resorption of type II and type III (Gustilo-Pasternak) compared with those with type-I loosening. The level of cytokines in joint fluid was not influenced by the time in situ of the implants or the age, gender or area of the osteolysis as measured on conventional radiographs. Our findings support the theory that macrophages in the joint capsule increase the production of TNF-α at an early phase probably because of particle load and in the absence of clinical loosening. Since TNF-α has an important role in the osteolytic process, the interfaces should be protected from penetration of joint fluid

Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al. 2. O. 3. and ZrO. 2. ) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 μm). Al. 2. O. 3. was compared with ZrO. 2. at 0.6 μm and with HDP at 4.5 μm. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al. 2. O. 3. particles. When comparing Al. 2. O. 3. and ZrO. 2. at 0.6 μm, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-α also increased with the size and concentration of Al. 2. O. 3. particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 μm and 350 particles per cell (p < 0.05). Release of TNF-α was higher with HDP than with Al. 2. O. 3. particles at 4.5 μm. However, we did not detect any significant difference in the release of TNF-α between Al. 2. O. 3. and ZrO. 2. at 0.6 μm (p > 0.05). We saw no evidence of release of interleukin-1α or interleukin-1ß after exposure to ceramic or HDP particles

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

Spontaneous muscle regenerative potential is limited, as severe injuries incompletely recover and result in chronic inflammation. Current therapies are restricted to conservative management, not providing a complete restitutio ad integrum; therefore, alternative therapeutic strategies are welcome, such as cell-based therapies with stem cells or Peripheral Blood Mononuclear Cells (PBMCs). Here, we described two different in vitro myogenic models: a 2D perfused system and a 3D bioengineered scaffold within a perfusion bioreactor. Both models were assembled with human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human primary skeletal myoblasts (hSkMs) to study induction and maintenance of myogenic phenotype in presence of PBMCs. When hBM-MSCs were cultured with human primary skeletal myoblasts (hSkMs) in medium supplemented with 10 ng/mL of bFGF; cells showed increased expression of myogenic-related gene, such as Desmin and Myosin Heavy Chain II (MYH2) after 21 days, and a prevalent expression of anti-inflammatory cytokines (IL10, 15-fold). Next, PBMCs were added in an upper transwell chamber and hBM-MSCs significantly upregulated myogenic genes throughout the culture period, while pro-inflammatory cytokines (e.g., IL12A) were downregulated. In 3D, hBM-MSCs plus hSkMs embedded in fibrin-based scaffolds, cultured in dynamic conditions, showed that all myogenic-related genes tended to be upregulated in the presence of PBMCs, and Desmin and MYH2 were also detected at protein level, while pro-inflammatory cytokine genes were significantly downregulated in the presence of PBMCs. In conclusion, our works suggest that hBM-MSCs have a versatile myogenic potential, enhanced and modulated by PMBCs. Moreover, our 3D biomimetic approach seemed to better resemble the tissue architecture allowing an efficient in vitro cellular cross-talk

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

Abstract. Introduction. Skeletal muscle wasting is an important clinical issue following acute traumatic injury, and can delay recovery and cause permanent functional disability particularly in the elderly. However, the fundamental mechanisms involved in trauma-induced muscle wasting remain poorly defined and therapeutic interventions are limited. Objectives. To characterise local and systemic mediators of skeletal muscle wasting in elderly patients following acute trauma. Methods. Experiments were approved by a local NHS Research Ethics Committee and all participants provided written informed consent. Vastus lateralis biopsies and serum samples were taken from human male and female patients shortly after acute trauma injury in lower limbs (n=6; mean age 78.7±4.4 y) and compared to age-matched controls (n=6; mean age 72.6±6.3 y). Atrogenes and upstream regulators (MuRF1; MAFbx; IL6, TNFα, PGC-1α) mRNA expression was assessed in muscle samples via RT-qPCR. Serum profiling of inflammatory markers (e.g. IL6, TNFα, IL1β) was further performed via multiplex assays. To determine whether systemic factors induced by trauma directly affect muscle phenotype, differentiated primary human myotubes were treated in vitro with serum from controls or trauma patients (pooled; n=3 each) in the final 24 hours of differentiation. Cells were then fixed, stained for myogenin and imaged to determine minimum ferret diameter. Statistical significance was determined at P<0.05. Results. There was an increase in skeletal muscle mRNA expression for E3 ligase MAFbx and inflammatory cytokine IL-6 (4.6 and 21.5-fold respectively; P<0.05) in trauma patients compared to controls. Expression of myogenic determination factor MyoD and regulator of mitochondrial biogenesis PGC-1α was lower in muscle of trauma patients vs controls (0.5 and 0.39-fold respectively; P<0.05). In serum, trauma patients showed increased concentrations of circulating pro-inflammatory cytokines IL-6 (14.5 vs. 0.3 pg/ml; P<0.05) and IL-16 (182.7 vs. 85.2 pg/ml; P<0.05) compared to controls. Primary myotube experiments revealed serum from trauma patients induced atrophy (32% decrease in diameter) compared to control serum-treated cells (P<0.001). Conclusion. Skeletal muscle from patients following acute trauma injury showed greater expression of atrophy and inflammatory markers. Trauma patient serum exhibited higher circulating pro-inflammatory cytokine concentrations. Primary human myotubes treated with serum from trauma patients showed significant atrophy compared to healthy serum-treated controls. We speculate a mechanism(s) acting via circulating factors may contribute to skeletal muscle pathology following acute trauma. 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

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development

Tendons mainly consist of collagen in order to withstand high tensile forces. Compared to other, high turnover tissues, cellularity and vascularity in tendons are low. Thus, the natural healing process of tendons takes long and can be problematic. In case of injury to the enthesis, the special transition from tendon over cartilage to bone is replaced by a fibrous scar tissue, which remains an unsolved problem in rotator cuff repair. To improve tendon healing, many different approaches have been described using scaffolds, stem cells, cytokines, blood products, gene therapy and others. Despite promising in vitro and in vivo results, translation to patient care is challenging. In clinics however, tendon auto- or allografts remain still first choice to augment tendon healing if needed. Therefore, it is important to understand natural tendon properties and natural tendon healing first. Like in other tissues, senescence of tenocytes seems to play an important role for tendon degeneration which is interestingly not age depended. Our in vivo healing studies have shown improved and accelerated healing by adding collagen type I, which is now used in clinics, for example for augmentation of rotator cuff repair. Certain cytokines, cells and scaffolds may further improve tendon healing but are not yet used routinely, mainly due to missing clinical data, regulatory issues and costs. In conclusion, the correct diagnosis and correct first line treatment of tendon injuries are important to avoid the necessity to biologically augment tendon healing. However, strategies to improve and accelerate tendon healing are still desirable. New treatment opportunities may arise with further advances in tendon engineering in the future