The collagenase of Clostridium Histolyticum enzyme infiltration is a mini-invasive treatment method for Dupuytren's disease which has emerged in recent years as an alternative to traditional surgery (selective aponeurectomy). Although both treatments are effective in the long term, a wider use of the enzyme is spreading worldwide. Indications and protocol of administration of collagenase are strictly regulated by the Italian Drug Administration Agency (AIFA). In the present study an off-label use of this medication has been experienced, in terms of wider indications and more numerous infiltration sites in the same cord (Multipoint technique) and in additional cords affecting other digits (Multicord technique). All patients suffering from Dupuytren's disease and accessing the Hand Surgery outpatient at Rizzoli Institute were considered for the study, between february 2014 and february 2016. Inclusion criteria were Dupuytren's disease and a positive tabletop test. The collagenase injection was indicated for degrees of passive extension deficit (PED) higher than AIFA regulations (MCPJoints >50° and PIPJoints >45°). These patients were compared with the same PED subgroup of surgical patients who were treated through aponeurectomy. Since the drug is dispensed in vials of 0.90 mg, but according to the protocol only 0.58 mg are to be infiltrated, the injection of the remaining 0.32 mg that would otherwise remain unused was experienced. Therefore, in patients who had only one pathological cord in the hand, the first point of the cord to be treated was inoculated with 0.58 mg, according to standards, while two additional points were selected along the fibrosis and injected with the remaining 0, 32 mg. This group was compared with patients treated with the traditional 0.58 mg only on a single cord. In patients in whom the presence of more than a single pathological cord was found, the worse lesion was injected with the usual 0.58 mg as by legislation and the second cord was infiltrated with the 0.32 mg residue and the results obtained within the second cord were compared with those achieved with the usual dose of 0.58 mg. The endpoints considered were the perioperative variations of passive extension deficit (PED) and range of motion (ROM), both expressed as degrees. Data were statistically analyzed in order to find any possible significance in the comparison of groups. Comparing the surgical patients with those treated with collagenase, for the same degrees but higher than AIFA reference, both methods showed a reduction of contracture by at least 50% at 30 days and an improvement of ROM (p>0.05), with fewer complications in those treated enzymatically (p<0.01). Infiltrating the whole dose of collagenase (0.90 mg) through the multipoint mode, has enabled an easier handling of the cord at 24 hours post-injection, a reduction in contracture of at least 50% at 30 days allowing a dowstaging of the disease and a better and faster recovery of hand function, than the classic treatment, although these results are not statistically significant (p>0.05). For degrees of contractures within AIFA indications for collagenase, the 0.32 mg dose is sufficient to cause the lysis of a cord with similar results compared to the greater AIFA-recommended dose of 0.58, in terms of all considered endpoints, with no statistically significant difference (p >0.01). This study confirms the success of treatment with collagenase compared to surgical treatment, in terms of efficacy, safety, more rapid recovery and less invasiveness. In addition, through further clinical studies, AIFA regulations can be gradually safely and effectively extended in terms of a progressive widening of indications and modalities including:. Indication to collagenase for PED higher than 50° (MCP joints) or 45° (PIP joints). Multiple injections in the same cord with the whole content of the vial (0.90 mg). Injections in multiple cords with the whole content of the vial (0.90 mg)

Xiapex is a novel non-surgical intervention for the treatment of Dupuytren's contracture. It comprises a fixed-ratio mixture of two purified collagenolytic enzymes (AUX-I [clostridial type I collegenase] and AUX II [clostridial type II collagenase]) isolated from the medium of Clostridium histolyticum. Xiapex targets the contracture through the injection of enzymes into the pathologic diseased cords.

The objectives of this study were to evaluate the safety and efficacy of Xiapex in patients with Dupuytren's Contracture, the recovery and associated use of health care resources. The study commenced in June 2011 and was completed in 11 months. 15 patients were screened for the study, 12 patients were eligible (10 male:2 female). All 12 patients completed the trial and 2 patients received multiple injections, one had two fingers affected and the other had two joints affected in one finger.

Following screening visit to determine patient suitability, the patient received their first injection usually within 7 days. Xiapex was injected directly into the palpable cord. 24 hours after injection patients were reviewed and a “finger extension procedure” was performed if the cord had not spontaneously ruptured. Patients were then followed up at 7 days, 30 days and 6 months. Assessments at each visits included: Finger Goniometry, Physician and Subject Global Assessment and Satisfaction, Health Care Resource Utilization and URAM (function assessment) and adverse events.

Of the 14 cords injected all ruptured after finger extension. No patient experienced any serious adverse events, but a number of more minor adverse events were recorded including skin tears, pain at injection site, swelling and bruising. At the time of final follow-up recurrence rates were 14%

In conclusion, Xiapex is suitable for appropriately selected patients who are affected by Dupuytren's contractures and who have a well-developed palpable cord.

Tendon-related pathologies such as tendinopathy represent a relevant clinical and socioeconomic issue. The most innovative and conservative therapeutic approaches are meant to stimulate the intrinsic healing capability of the tissue. In this study, the use of pulsed electromagnetic fields (PEMFs) was investigated in a rat model of Achilles tendinopathy as a potential therapy. Achilles tendinopathy was chemically induced in eighty-six Sprague Dawley rats by injecting collagenase Type I within the tendon fibers. Fifty-six of them were stimulated with PEMFs (8 hours/day, 1.5 ± 0.2 mT; 75 Hz), divided in different experimental groups basing on the starting-time of PEMFs exposure (after 0, 7, 15 after Collagenase injection) and its duration (7, 15 or 30 days). Thirty animals were left unstimulated (CTRL group). According to the different time points, explanted tendons were evaluated through histological and immunohistochemical analyses in term of matrix deposition, fiber re-organization, neovascularization and inflammatory reaction. The most effective PEMF stimulation was demonstrated in the 15 days of treatment. However, when PEMF were applied immediately after the collagenase injection, no significant therapeutic results were found. On the contrary, when PEMF were applied after 7 and 15 days from the collagenase injection, they promoted the deposition of extracellular matrix and tendon fiber re-organization, reducing both the inflammatory reaction and vascularization, with significant differences compared to the CTRL group (p<0.05). Therefore, these results suggest an effective activity of PEMFs stimulation that provides a satisfying restoration of the damaged tissue, although the most performing protocol of application still needs to be identified

Introduction and Objective. Guided Bone Regeneration (GBR) uses biodegradable collagen membranes of animal origin tissues (dermis and pericardium). Their barrier effect prevents soft tissues to interfere with the regeneration of alveolar bone. However, their xenogeneic origin involves heavy chemical treatments which impact their bioactivity. Wharton's Jelly (WJ) from the umbilical cord is a recoverable surgery waste. WJ is mostly made from collagen fibers, proteoglycans, hyaluronic acid, and growth factors. WJ with immunologically privileged status and bioactive properties lends credence to its use as an allograft. Nevertheless, low mechanical properties limit its use in bone regenerative strategies. Herein, our objective is to develop a crosslinked WJ-based membrane to improve its strength and thus its potential use as a GBR membrane. Materials and Methods. The umbilical cords are collected after delivery and then stored at −20°C until use. The WJ membranes (1 × 5 × 12 mm) were obtained after the removal of blood vessels and amniotic tissue, washed, lyophilized, and stored at −20°C. WJ membranes were incubated in genipin solutions in decreasing concentrations (0.3 g / 100 mL − 0.03 g / 100 mL) for 24 hours at 37°C. The crosslinking degree was estimated by ninhydrin and confirmed by FTIR (Fourier-transform infrared spectroscopy) assays. The swelling rate was obtained after the rehydration of dry crosslinked WJ-membrane for 10 min in D-PBS. The mechanical properties were assessed in hydrated conditions on a tensile bench. The resistance to the degradation was evaluated by collagenase digestion (1 mg/mL for 60 hours) assay. The cytotoxicity of crosslinked WJ-membrane was evaluated in accordance with the standard ISO.10993-5 (i.e. Mitochondrial activity and Lactate Dehydrogenase release) against Mesenchymal Stem Cells (MSCs). Finally, the MSCs colonization and proliferation were followed after 21 days of culture on crosslinked WJ-membranes. Results. The increase of crosslinking rates from 30% to 90% of the WJ membrane was demonstrated by the ninhydrin assay. FTIR analysis showed a prominent peak at 1732 cm. -1. , confirming the incorporation of genipin in the WJ. The swelling rate of crosslinked WJ-membrane decreased with an increase of the crosslinking rate. An increase in elastic modulus and an increase in the resistance to the collagenase degradation were observed along with an increase in the crosslinking degree. Cytotoxicity investigations did not elicit a harmful effect of the genipin, however, a poor MSCs adhesion on the crosslinked membrane was observed. Conclusions. Our results show that a membrane can be developed from Wharton's jelly. The mechanical and degradation properties can be improved by crosslinking with genipin without inducing any cytotoxicity effect. However, the percentage of crosslinking has an influence on the adhesion of the cells to the membranes. The crosslinked WJ-membrane bioactivity and the osteo-regenerative potential in vitro/in vivo will be evaluate

Introduction and Objective. Osteoarthritis (OA) represents one of the leading cause of disability all over the world. Cell therapies, mainly based on mesenchymal stem cells (MSCs), have shown to modulate the pathogenesis of OA in basic, preclinical and clinical studies. Adipose tissue (AT) have emerged as a rich and promising source of MSCs called adipose derived stem cells (ASCs). Different systems are available for processing lipoaspirate to purify the samples from oily and haemorrhagic fractions, minimizing the risk of complications and maximizing the biological yield for subsequent grafting. However, few studies compared the efficacy of the different processing devices already used in clinical practice. This study aims to characterize the products obtained by the use of two different systems such as micro-fragmentation or nano-fragmentation comparing them with the starting material (AT) and the collagenase isolated ASCs. Materials and Methods. AT from 12 donors arrived without selection to the laboratories: 4 lipoaspirated (LA), 4 micro-fragmented (mF) and 4 nano-fragmented (nF). The samples were divided into three aliquots for paraffin embedding, RNA extraction and digestion with collagenase for ASCs isolation. Paraffin embedded tissue sections were stained with hematoxylin-eosin to analyze morphology. RNA was extracted, retro-transcribed and analyzed with real-time PCR to analyze the expression of pluripotency genes (SOX2, NANOG and POU5F1) and inflammatory genes (IL-1beta and iNOS). Data were analyzed using Graphpad Prism 8.0 and expressed as mean ± SD. One-way ANOVA followed by Tukey test was used to compare the different groups. Results. The LA comprised small lobules, with intact cell membranes and structurally integer adipocytes. mF samples showed the presence of integer adipocytes, small lobules and higher amount of cell clusters. nF samples showed the almost completely absence of adipocytes, a high amount of cells without lipid content and a high amount of stromal matrix. Real-time PCR results showed the lowest expression levels of pluripotency genes in LA samples that were assumed equal to 1.0 and used to calculate the expression levels of the other samples. mF showed expression levels of pluripotency genes similar to AT. nF showed expression levels of pluripotency genes higher than AT and mF, but without statistically significant differences. ASCs showed statistically significant higher expression levels of these genes compared to LA and mF (p ≤ 0.001). Likewise, the expression of inflammatory genes resulted to be lowest in LA samples (assumed equal to 1.0), higher in mF samples and in nF samples without statistical significance. As expected, the highest values were found in ASCs isolated cells compared to all the other samples (p ≤ 0.0001). Conclusions. These results confirmed that micro-fragmentation (mF) and nano-fragmentation (nF) permitted to separate a cell mixture enriched in ASCs from a lipoaspirate sample without activating the inflammatory pathways. Both processing methods gave a minimally manipulated product suitable for OA cell therapy application. Further studies are needed to elucidate possible different activities of the ASCs enriched AT-derivatives

Objectives. Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA. Methods. Collagenase (type II) was injected twice into both knees of three-month-old rabbits to induce OA, combined with two intra–articular injections of Torin 1 (400 nM). Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at eight weeks after injection. Chondrocyte degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13 (MMP-13) and vascular endothelial growth factor (VEGF) expression were analysed by quantitative RT-PCR (qPCR).Beclin-1 and light chain 3 (LC3) expression were examined by Western blotting. Results. Intra-articular injection of Torin 1 significantly reduced degeneration of the articular cartilage after induction of OA. Autophagosomes andBeclin-1 and LC3 expression were increased in the chondrocytes from Torin 1-treated rabbits. Torin 1 treatment also reduced MMP-13 and VEGF expression at eight weeks after collagenase injection. Conclusion. Our results demonstrate that intra-articular injection of Torin 1 reduces degeneration of articular cartilage in collagenase-induced OA, at least partially by autophagy activation, suggesting a novel therapeutic approach for preventing cartilage degeneration and treating OA. Cite this article: N-T. Cheng, A. Guo, Y-P. Cui. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model. Bone Joint Res 2016;5:218–224. DOI: 10.1302/2046-3758.56.BJR-2015-0001

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

Depletion of Scleraxis-lineage (ScxLin) cells in adult tendon recapitulates age-related decrements in cell density, ECM organization and composition. However, depletion of ScxLin cells improves tendon healing, relative to age-matched wildtype mice, while aging impairs healing. Therefore, we examined whether ScxLin depletion and aging result in comparable shifts in the tendon cell environment and defined the intrinsic programmatic shifts that occur with natural aging, to define the key regulators of age-related healing deficits. ScxLin cells were depleted in 3M-old Scx-Cre+; Rosa-DTRF/+ mice via diphtheria toxin injections into the hindpaw. Rosa-DTRF/+ mice were used as wildtype (WT) controls. Tendons were harvested from 6M-old ScxLin depleted and WT mice, and 21-month-old (21M) C57Bl/6 mice (aged). FDL tendons (n=6) were harvested for single-cell RNAseq, pooled, collagenase digested, and sorted for single cell capture. Data was processed using Cell Ranger and then aligned to the annotated mouse genome (mm10). Filtering, unsupervised cell clustering, and differential gene expression (DEG) analysis were performed using Seurat. Following integration and sub-clustering of the tenocyte populations, five distinct subpopulations were observed. In both ScxLin depletion and aging, ‘ECM synthesizers’ and ‘ECM organizers’ populations were lost, consistent with disruptions in tissue homeostasis and altered ECM composition. However, in ScxLin depleted mice retention of a ‘specialized ECM remodeler’ population was observed, while aging tendon cells demonstrated inflammatory skewing with retention of a ‘pro-inflammatory tenocyte population’. In addition, enrichment of genes associated with protein misfolding clearance were observed in aged tenocytes. Finally, a similar inflammatory skewing was observed in aged tendon-resident macrophages, with this skewing not observed in ScxLin depleted tendons. These data suggest that loss of ‘ECM synthesizer’ populations underpins disruptions in tendon homeostasis. However, retention of ‘specialized remodelers’ promotes enhanced healing (ScxLin depletion), while inflammatory skewing may drive the impaired healing response in aged tendons

Summary:. Hamstring tendons (HT) represent a widely used autograft for ACL reconstruction. Harvesting, processing and pretensioning procedures together with the time out of the joint could theoretically hamper tendon cells (TCs) viability. The authors hypothesize that HT cells are not impaired at the end of the surgical procedures and their tenogenic phenotype may be strongly improved by exposure to PEMF. Methods. Remnants of semitendinosus and gracilis tendons were collected at the end of the surgical procedures before skin closure from 15 healthy donors who underwent ACL reconstruction with autologous hamstring tendons. To isolate TCs, the tendon was minced and digested with 0.3 % type I collagenase and the nucleated cells were plated at a density 5x10E3 cells/cm2 and cultured in chamber slides in differentiation medium composed of DMEM + 5ng/ml basic fibroblast growth factor (b-FGF) for 7, 14, 21 days. The following cell cultures were set up:. -. TCs cultured with differentiation medium + exposure to PEMF 8 h/day (PEMF generator system IGEA, intensity of magnetic field = 1.5 mT, frequency = 75 Hz). -. TCs cultured with differentiation medium without exposure to PEMF. At day 0, day 7, day 14 and day 21, immunofluorescence analysis was performed to evaluate the expression of collagen type I, collagen type VI, scleraxis and PCNA (proliferative marker). Subsequently, tendon explant cultures were set up to verify, at day 21, explant viability and the expression of collagen type I, collagen type VI, beta-catenin and PCNA. Results. The TCs from the tendon fragments at the end of the ACL reconstruction were alive and they expressed markers of proliferation and tendon phenotype at the end of the culture periods. The TCs in the presence PEMF 8h/day showed greater production of collagen type I, collagen type VI and scleraxis than that of TCs cultured without PEMF (p<0,05): the expression of this markers increased from 7 to 21 days of culture. The expression of PCNA, in the presence of PEMF stimulus, was significantly lower (p<0,05) than that of TCs cultured without PEMF. A similar behavior was surprisingly observed in tendon explant cultures. Conclusions. Hamstring tendons used for ACL reconstruction are not simple autologous tenoconductive scaffold but are a biologic structure rich in progenitor cells that show tenogenic behavior. Their tenogenic phenotype may be strongly improved by exposure to PEMF. In a future clinical perspective, the postoperative use of PEMF could be used to enhance the ligamentization processes of autologous hamstring tendons, when used as autografts for ACL reconstructions

Abstract. Objectives. The enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production. Methods. Cell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×10. 4. , 5×10. 4. , 1×10. 5. , 2×10. 5. in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope. 3D mini-tissue constructs. 15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix. Results. RTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×10. 5. RTFs and 3×10. 5. BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage. Conclusions. This study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction and Objective. Intervertebral disc (IVD) degeneration accompanying with low back pain is a serious worldwide problem. Even though, surgical treatments are available for pain relief, there is an urgent need to establish enduring cell-based remedies. Notochordal (NC) cells as the ancestor of nucleus pulposus (NP) cells in human IVD are a promising therapeutic target. It has been reported that the loss of NC cells after childhood could promote the onset of disc degeneration. Thus, we firstly, aimed to optimise the culture of NC cells in vitro without using the FCS in alginate (3D) culture systems, secondly, investigate their behaviour in healthy and degenerate niche and lastly, co-culture these cells with degenerated NP cells to assess their regeneration potentials. Materials and Methods. Porcine NC cells were extracted using pronase treatment followed by overnight digestion in 0.01% collagenase II. After extraction, cells were culture in 1.2% alginate beads (gold standard 3D culture) in either low glucose DMEM or αMEM medium. Cells were harvested after 24 hours, 1 week and 2 weeks for gene expression analysis and formalin fixed paraffin embedding. Quantitative Real-Time PCR and Immuno-staining were performed for analysis of NC markers (KRT18, FOXA2 and T) and COL I as a negative marker. Next, NC cells were cultured in healthy and degenerate medium to assess their viability and behaviour. Results. A mixed phenotype of NC and NP cells was observed in alginate bead cultures. NC phenotype was observed within all culture conditions with production of GAGs and maintenance of vacuolated phenotype. Gene expression analysis showed no significant difference between the culture of NC cells in low glucose DMEM and αMEM medium. Interestingly, NC cell viability was maintained in both healthy and degenerate media, despite observing more dead cells in degenerate conditions. Current investigations are comparing the behaviour of NC cells in healthy and degenerate niche. Conclusions. Investigating the preservation of NC phenotype in alginate culture and studying their behaviour between healthy and degenerate conditions would lead us to better understand their characteristics in different niches and how we can further use them in therapeutic purposes for disc degeneration

Introduction and Objective. The meniscus is composed of two distinct regions, a vascular outer zone and an avascular inner zone. Due to vascularization, tears within the vascular zone can be treated by suturing. However, tears in the avascular zone have a poor healing capacity and partial meniscectomy is used to prevent further pain, although this leads to early osteoarthritis. Previous studies have demonstrated that the vascular zone contains a progenitor population with multilineage differentiation potential. Isolation and propagation of these progenitors can be used to develop cell-based therapies for treating meniscal defects. In vivo, the meniscus resides under a low oxygen environment, also known as physioxia (2–7% oxygen) and previous work suggests that it promotes the meniscal phenotype. The objective of the study was to isolate progenitor populations from both meniscus regions and to examine their clonogenecity and differentiation potential under both hyperoxia (20% oxygen) and physioxia (2% oxygen). We hypothesize that physioxia will have a beneficial effect on colony formation and trilineage differentiation of meniscal cells. Materials and Methods. Human meniscus (n =4; mean age: 64 + 6) tissue was split into vascular and avascular regions, finely cut into small pieces and then sequentially digested in pronase (70U/mL) and collagenase (200U/mL) at 37. 0. C. Avascular and vascular meniscus cells were counted and split equally for expansion under hyperoxia and physioxia at a seeding density of 5 × 10. 3. cells/cm. 2. At passage 1, cells were seeded at 2, 5 and 20 cells/cm. 2. in 10cm dishes for observing colony formation using crystal violet assay. At passage 3, vascular and avascular meniscus cells were differentiated towards the chondrogenic, osteogenic and adipogenic lineage. Chondrogenesis was evaluated using DMMB staining for GAG deposition, osteogenesis was assessed using Alizarin Red staining for calcium deposition, whilst adipogenesis was observed using Oil-Red-O staining for fat droplets. Results. Expansion of vascular and avascular meniscus cells showed no difference in doubling time between hyperoxic or physioxic culture. However, physioxia significantly increased the number of colonies compared to hyperoxia for both meniscus cell types (p < 0.05). Both vascular and avascular meniscus cells differentiated towards the chondrogenic, osteogenic and adipogenic lineage under both oxygen tensions. Interestingly, we observed greater DMMB, alizarin red and oil-red-o staining for vascular meniscal cells under physioxia compared to corresponding hyperoxic cultures and avascular meniscal cells. Conclusions. Physioxia enhances the clonogenecity of vascular and avascular meniscus cells. Trilineage differentiation potential was observed from both regions with increased capacity detected under physioxia for vascular meniscal cells. Physioxic isolation of meniscal cells for the propagation of these progenitors can used be for the treatment of meniscal tears/defects

Introduction and Objective. Low back pain (LBP) is a disorder strongly associated with intervertebral disc degeneration (IDD) with an important impact on the quality of life of affected people. To date, LBP treatment is based on conservative methods with the aim to reduce back pain without restoring the degenerative environment of the disc. The main cause of IDD is the drastic reduction of the proteoglycan content within the nucleus pulposus (NP), eventually leading to the loss of disc water content, micro-architecture, biochemical and mechanical properties. A promising approach for disc regeneration is represented by the transplantation of mesenchymal stromal cells (MSCs). The exact mechanism remains unknown. Growing evidence suggests that MSCs can influence cells and modulate cells’ behaviour by secreting a set of bioactive factors. MSCs secretome is composed of several molecules such as soluble protein, lipids, nucleic acids and extracellular vesicles (EVs) involved in inflammation, immunomodulation, cell survival and intercellular communication. The aim of this study was to evaluate the in vitro effects of MSCs secretome on human NP cells (hNPCs) in a 3D culture model with and without inflammatory stimulus. Materials and Methods. MSCs secretome was collected from bone marrow-MSCs (BM-MSCs) and adipose tissue-MSCs (ASCs) after centrifugation and obtained by culturing cells without fetal bovine serum (FBS) for 48 hours. hNPCs were isolated from surgical specimens through digestion with type II collagenase, culture expanded in vitro, encapsulated in alginate beads (three-dimensional culture system) and treated with growth medium (controls), BM-MSCs or ASCs secretome with or without interleukin-1 beta (IL-1b). After 7 days, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). Cell proliferation and glycosaminoglycan (GAG) production was assessed by flow cytometry and 1,9-dimethylmethylene blue (DMMB), respectively. hNPCs in alginate beads were stained with Live/Dead assay and detected using confocal immunofluorescence microscopy. Data were analyzed using Graphpad prism 8 and expressed as mean ± S.D. One-way ANOVA analysis was used to compare differences among the groups under exam. Results. Our results reported an increase of hNPCs proliferation after treatment with both MSCs-secretomes. In detail, cell proliferation levels increased at 7 days after ASC-secretome (p ≤ 0,05) and BM-secretome (p ≥ 0,05) treatment compared to control. Live/dead staining showed that cell death was reduced by BM-secretome (p ≤ 0,05); in combined treatment of BM-secretome with IL1b 10ng/mL (p ≤ 0,05) at 7 days compared to control. There is not a significant difference between treated and untreated hNPCs’ GAG synthesis. In addition, gene expression levels resulted to be modulated by MSCs-secretomes under study compared to controls. Conclusions. Although the cell-therapy may be considered an attractive and safe option, MSCs require long and expensive processes. In conclusion, our experimental conditions supported as BM-MSCs and ASCs secretomes could represent cell-free alternative approaches in IDD, overcoming translational limits of cell therapy to the clinical practice

Introduction and Objective. Anterior cruciate ligament reconstruction (ACLR) with tendon autografts is the “gold standard” technique for surgical treatment of ACL injuries. Common tendon graft choices include patellar tendon (PT), semitendinosus/gracilis “hamstring” tendon (HT), or quadriceps tendon (QT). Healing of the graft after ACLR may be affected by graft type since the tissue is subjected to mechanical stresses during post-operative rehabilitation that play important roles in graft integration, remodeling and maturation. Abnormal mechanical loading can result in high inflammatory and degradative processes and altered extracellular matrix (ECM) synthesis and remodeling, potentially modifying tissue structure, composition, and function. Because of the importance of load and ligamentization for tendon autografts, this study was designed to compare the differential inflammatory and degradative metabolic responses to loading by three tendon types commonly used for autograft ACL reconstruction. Materials and Methods. With IRB approval (IRB # 2009879) and informed patient consent, portions of 9 QT, 7 PT and 6 HT were recovered at the time of standard of care ACLR surgeries. Tissues were minced and digested in 0.2 mg/ml collagenase solution for two hours and were then cultured in 10% FBS at 5% CO. 2. , 37°C, and 95% humidity. Once confluent, cells were plated in Collagen Type I-coated BioFlex® plates (1 × 10. 5. cells/well) and cultured for 2 days prior to the application of strain. Then, media was changed to supplemented DMEM with 2% FBS for the application of strain. Fibroblasts were subjected to continuous mechanical stimulation (2-s strain and 10-s relaxation at a 0.5 Hz frequency) at three different elongation strains (mechanical stress deprivation-0%, physiologic strain-4%, and supraphysiological strain-10%). 9. for 6 days using the Flexcell FX-4000T strain system. Media was tested for inflammatory biomarkers (PGE2, IL-8, Gro-α, and MCP-1) and degradation biomarkers (GAG content, MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2). Significant (p<0.05) difference between graft sources were assessed with Kruskal-Wallis test and post-hoc analysis. Results are reported as median± interquartile range (IQR). Results. Differences in Inflammation-Related Biomarker Production (Figure 1): The production of PGE2 was significantly lower by HT fibroblasts compared to both QT and PT fibroblasts at all timepoints and strain levels. The production of Gro-α was significantly lower by HT fibroblasts compared to QT at all time points and strain levels, and significantly lower than PT on day 3 at 0% strain, and all strain levels on day 6. The production of IL-8 by PT fibroblasts was significantly lower than QT and HT fibroblast on day 3 at 10% strain. Differences in Degradation-Related Biomarker Production (Figure 2): The production of GAG by HT fibroblasts was significantly higher compared to both QT and PT fibroblasts on day 6 at 0% strain. The production of MMP-1 by the QT fibroblasts was significantly higher compared to HT fibroblasts on day 3 of culture at all strain levels, and in the 0% and 10% strain levels on day 6 of culture. The production of MMP-1 by the QT fibroblasts was significantly higher compared to PT fibroblasts at in the 0% and 4% strain groups on day 3 of culture. The production of TIMP-1 by the HT fibroblasts was significantly lower compared to PT fibroblasts on day 3 of culture. Conclusions. The results of this study identify potentially clinically relevant difference in the metabolic responses of tendon graft fibroblasts to strain, suggesting a lower inflammatory response by hamstring tendon fibroblasts and higher degradative response by quadriceps tendon fibroblasts. These responses may influence ACL autograft healing as well as inflammatory mediators of pain in the knee after reconstruction, which may have implications regarding graft choice and design of postoperative rehabilitation protocols for optimizing outcomes for patients undergoing ACL reconstruction. For any figures or tables, please contact the authors directly

Tendinopathies represent the 45% of the musculoskeletal lesions and they are a big burden in clinics. Indeed, despite the relevant social impact, both the pathogenesis and the development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. Indeed, current treatment for tendinopathy are mainly symptomatic, and they present a high rate of pathology re-occurrence. In this contest, the development of an efficient in vivo model of acute tendinopathy, focused on the choice of the most appropriate species and strategy to induce the disease, would allow a better understanding of the pathology progression throughout its phases. Then, the purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies. 40 Sprague Dawley rats were randomly divided into two groups, treated by injection of collagenase type I within the Achilles tendon at 1 mg/mL (low dose, LD) or 3 mg/mL (high dose, HD). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days by H&E staining. Our results showed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater fatty degeneration and neovascularization with respect to the lower dose. These changes are time-dependent, thus resembling the tendinopathy development in humans. Indeed, the acute phase occurred from day 3 to day 15, while from day 15 to 45 it progressed towards the proliferative phase, displaying a degenerative appearance associated with a precocious remodeling process. The model represents a good balance between feasibility, in terms of reproducibility and costs, and similarity with the human disease. Moreover, the present model contributes to improve the knowledge about tendinopathy development, and then it could be useful to design further pre-clinical studies, in particular in order to test innovative treatments for tendinopathy

Intervertebral disc degeneration (IDD) affects more than 80% of the population and is often linked to a reduction of the proteoglycan content within the nucleus pulposus (NP). The nutritional decline and accumulation of degraded matrix products promote the inflammatory process favoring the onset of disease. Several regenerative approaches based on cell therapy have been explored. Recently, paracrine factors and extracellular vesicles (EVs) such as exosomes have been described to play a fundamental role in the cross-talk between mesenchymal stem cells (MSCs) and NP in the microenvironment. EVs vehicule different molecules: proteins, nucleic acids and lipids involved in intercellular communication regulating the homeostasis of recipient cells. Therefore, MSCs-derived exosomes are an interesting emerging tool for cell-free therapies in IDD. The aim of this study was to evaluate the in vitro effects of MSCs derived exosomes on human NP cells (hNPCs). Exosomes were isolated through a multistep ultracentrifugation of bone marrow-MSCs (BM-MSCs) conditioned media (CM), obtained by culturing BM-MSCs without fetal bovine serum (FBS) for 48 hours. Exosomal morphology was characterized by transmission electron microscope (TEM). The exosomes were quantified by bicinchoninic acid assay (BCA) and cryopreserved at –80 °C. hNPCs derived from surgical speciments digested with type II collagenase. After culture expansion in vitro, hNPCs in alginate beads (three-dimensional culture system) were treated with growth medium (controls), exosomes, CM, interleukin-1 beta (IL-1b), IL-1b plus exosomes, IL-1b plus CM. After 24 hours, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). TEM analysis confirmed the cup-shaped vescicles in our preparations. Gene expression levels resulted to be modulated by both exosomes and CM compared to controls. In addition, both treatments were capable to alter the inflammatory stimuli of IL-1b. Interestingly, exosomes were able to change anabolic and catabolic gene expression levels differently from CM. In our experimental conditions, both exosomes and CM from BM-MSCs could be an interesting alternative strategy in intervertebral disc regeneration, overcoming the costs and translational limits of cell therapy to the clinical practice

Uncemented implants combining antimicrobial properties with osteoconductivity would be highly desirable in revision surgery due to periprosthetic joint infection (PJI). Silver coatings convey antibacterial properties, however, at the cost of toxicity towards osteoblasts. On the other hand, topological modifications such as increased surface roughness or porosity support osseointregation but simultaneously lead to enhanced bacterial colonization. In this study, we investigated the antibacterial and osteoconductive properties of silver-coated porous titanium (Ti) alloys manufactured by electron beam melting, rendering a macrostructure that mimics trabecular bone. Trabecular implants with silver coating (TR-Ag) or without coating (TR) were compared to grit-blasted Ti6Al4V (GB) and glass cover slips as internal controls. Physicochemical characterization was performed by X-ray diffraction (XRD) and energy dispersive X-rays (EDX) together with morphological characterization through electron scanning microscopy (SEM). Bacterial adherence after incubation of samples with Staphylococcus (S.) aureus and S. epidermidis strains harvested from PJI patients was quantitatively assessed by viable count after detachment of adherent bacteria by collagenase/dispase treatment. Primary human osteoblasts (hOB) were used to investigate the osteoconductive potential by lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. Cell morphology was investigated by fluorescence microscopy after staining with carboxifluorescein diacetate succinimidyl ester (CFDA-SE) and 4′,6-diamidino-2-phenylindole (DAPI). The trabecular implants depicted a porosity of 70% with pore sizes of 600µm. The amount of silver analyzed by EDX accounted for 35%wt in TR-Ag but nil in TR. Silver-coated TR-Ag implants had 24% lower S. aureus viable counts compared to non-coated TR analogues, and 9% lower compared to GB controls. Despite trabecular implants, both with and without silver, had higher viable counts than GB, the viable count of S. epidermidis was 42% lower on TR-Ag compared to TR. The percentage of viable hOB, measured by LDH and normalized to controls and area at 1 day, was lower on both TR-Ag (18%) and on TR (13%) when compared with GB (89%). However, after 1 week, cell proliferation increased more markedly on trabecular implants, with a 5-fold increase on TR-Ag, a 3.4-fold increase on TR, and a 1.7-fold increase on GB. Furthermore, after 2 weeks of hOB culture, proliferation increased 20-fold on TR-Ag, 29-fold on TR, and 3.9-fold for GB, compared to 1 day. The osteoconductive potential measured by ALP illustrated slightly higher values for TR-Ag compared to TR at 1 day and 2 weeks, however below those of GB samples. Cell morphology assessed by microscopy showed abundant growth of osteoblast-like cells confined to the pores of TR-Ag and TR. Overall, our findings indicate that the silver coating of trabecular titanium exerts limited cytotoxic effects on osteoblasts and confers antimicrobial effects on two PJI-relevant bacterial strains. We conclude that improving material design by mimicking the porosity and architecture of cancellous bone can enhance osteoconductivity while the deposition of silver confers potent antimicrobial properties

Human mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate into mesoderm-type cells such as osteoblasts, chondroblast, tenocytes etc. They can be retrieved by different sources, but the number of cells obtained suggested the adipose tissue as a primary harvest site of MSCs. Cells can be harvested using the Coleman procedure, obtaining stromal vascular fraction (SVF), enriched with MSCs, after collagenase digestion. The availability of SVF storage has been envisioned for multiple treatments of the degenerated tissue. Indeed, the use of SVF has been introduced into clinical trials for tissue regeneration for orthopaedic patients. Difficulties of a selective delivery of SVF locally have been previously discussed. Thus, the use of biological scaffolds in order to better localize SVF in the tissue site has been studied. The methodological evolution for the use of SVF in the best possible biological conditions is a milestone for good clinical results

Complex pathophysiologies involve different signalling mechanisms, with a multitude of often interconnected potential therapeutic targets. Therefore, there is a need for the development of multi-compartment delivery vehicles for combinatorial and synergistic therapeutic approaches. In this study it was hypothesized that multi-compartment crosslinked collagen type I systems can deliver multiple bioactive agents in a controlled manner in an in vitro model condition of skin fibrosis. Multi-compartment collagen-based systems were made using solutions of dialyzed type I collagen mixed with 10× PBS, after which they were neutralised and crosslinked with 1 and 2.0 mM 4 arm-succinimidyl glutarate ester PEG (4 arm-PEG-SG), respectively, followed by incubation at 37ºC. The systems were characterised through swelling assessment, collagenase degradation assay and compression tests. The release of encapsulated drugs from the hydrogels was studied by ELISA and the effect of the delivered bioactive agents was assessed through imaging and quantification for fibrotic markers in an in vitro model. A pilot study using FITC-dextran proved that the inner compartment was capable of promoting a sustained release over a long period of time (7 days), which was further confirmed with drug release assays using a TNF-α antagonist and recombinant decorin, fitting the intended therapeutic release profile. Protein expression studies showed a decrease of endogenous collagen type I and α-smooth muscle actin expression (p<0.05) indicating amelioration of fibrosis. In summary, this indicates that this system is suitable for dual delivery of multiple bioactive agents, resulting in a controlled release in vitro and illustrating its potential in therapy

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans