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Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043—a derivative of angiopoietin-like 3 (ANGPTL3)—as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5β1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial (NCT02491281; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial (NCT04864392) in patients with knee OA.

Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft&reg, ) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft&reg, This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.

Engineered hypertrophic cartilage (HC) represents an attractive bone substitute material, capable to induce bone formation by endochondral ossification. Since bone formation by HC depends on factors released from the extracellular matrix, in this study, we hypothesized that HC seeding with monocytes committed to osteoclastogenesis could enhance its remodeling, improve chemotaxis of skeletal and vascular cells, and consequently enhance bone formation. This would be particularly relevant for devitalized HC, which currently exhibits only limited osteoinductivity. Living or devitalized HC engineered from human bone marrow-derived mesenchymal stromal cells (MSCs) was seeded or not with human monocytes in the presence of macrophage colony-stimulating factor and RANK-ligand, cultured for up to 15 days, or implanted ectopically in nude mice. Monocytes seeded on devitalized, but not living, HC induced its in vitro resorption, resulting in 30-fold higher release and 2.7-fold lower content of glycosaminoglycans compared with unseeded samples. In vitro, supernatants from monocyte-seeded devitalized HC attracted more monocytes compared with unseeded samples, but did not enhance chemotaxis of MSCs or human umbilical vein endothelial cells. In vivo, however, neither remodeling nor invasion by osteoclasts, endothelial cells, and mouse MSCs were significantly affected by the seeding with monocytes. Finally, in vitro priming of living or devitalized HC by monocytes did not enhance their bone-forming capacity. Further investigations should test the proposed approach on HC engineered to prevent rapid degradation and support osteoclastogenesis, or identify alternative strategies to enhance engineered HC remodeling and bone-forming capacity.

Abstract Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue—which include mesenchymal, endothelial, and osteoclastic progenitors—enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells. Significance This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals.

Cartilage repair/regeneration procedures (e.g., microfracture, autologous chondrocyte implantation [ACI]) typically result in a satisfactory outcome in selected patients. However, the vast majority of patients with chronic symptoms and, in general, a more diseased joint, do not benefit from these surgical techniques. The aims of this work were to (1) review factors negatively influencing the joint environment; (2) review current adjuvant therapies that can be used to improve results of cartilage repair/regeneration procedures in patients with more diseased joints, (3) outline future lines of research and promising experimental approaches. Chronicity of symptoms and advancing patient age appear to be the most relevant factors negatively affecting clinical outcome of cartilage repair/regeneration. Preliminary experience with hyaluronic acid, platelet-rich plasma, and mesenchymal stem cell has been positive but there is no strong evidence supporting the use of these products and this requires further assessment with high-quality, prospective clinical trials. The use of a Tissue Therapy strategy, based on more mature engineered tissues, holds promise to tackle limitations of standard ACI procedures. Current research has highlighted the need for more targeted therapies, and (1) induction of tolerance with granulocyte colony-stimulating factor (G-CSF) or by preventing IL-6 downregulation; (2) combined IL-4 and IL-10 local release; and (3) selective activation of the prostaglandin E2 (PGE2) signaling appear to be the most promising innovative strategies. For older patients and for those with chronic symptoms, adjuvant therapies are needed in combination with microfracture and ACI.

Purpose The aim of this study is to prospectively evaluate the medium-term effectiveness and regenerative capability of autologous adult mesenchymal stem cells, harvested as bone marrow aspirate concentrate (BMAC), along with a hyaluronan-based scaffold (Hyalofast) in the treatment of ICRS grade 4 chondral lesions of the knee joint, in patients older than 45 years. Methods A study group of 20 patients with an age >45 years (mean 50.0 ± 4.1 years) was compared to a control group of 20 patients with an age 45 years population with functional outcomes that are comparable to younger patients at final follow-up. Level of evidence Prospective cohort study, Level II.

Subclavius posticus muscle is a supernumerary anatomical variation of the subclavius muscle. The aim of this study was to show the possible contribution of the posticus muscle in the development of unilateral thoracic outlet syndrome (TOS) in overhead athletes, presenting hypertrophy of the dominant arm due to their sport activity. Reported here are 2 young overhead athletes complaining pain, paresthesia, weakness in the dominant upper limb, although presenting none of the main shoulder and neurological disorders. After developing subclavian vein thrombosis, TOS was suspected and finally diagnosed by dynamic magnetic resonance angiography, which also showed bilateral subclavius posticus muscle in both patients. Despite bilateral subclavius posticus, TOS was only evident in the dominant limb in which the association of hypertrophy of lateral cervical muscles, resulting from the overhead sport activity, to the subclavius posticus likely led to a significant reduction of the upper thoracic outlet space.

OBJECTIVE: To investigate T(2) mapping as a possible marker for low-grade human articular cartilage lesions during a one-year follow-up, possible changes during the follow-up and compare the reliability and sensitivity of these measurements on high-field (3 T) and ultra-high-field (7 T) MRI scanners. DESIGN: Twenty-one patients with femoral, tibial and patellar cartilage defect in the knee joint participated in the study. The MRI protocol consisted of morphological, as well as three-dimensional triple-echo steady-state (3D-TESS) T(2) mapping sequences with similar parameters at 3T and 7T. Patients were scanned at five time-points up to 12 months. T(2) values were evaluated in the lesion and healthy-appearing regions for superficial and deep cartilage zone. The repeated ANOVA was used to determine differences in T(2) values at various time points. RESULTS: A significant decrease in T(2) values was observed between baseline and six months in the superficial layer of the lesion in patients at 3 T (decrease from 41.89 ± 9.3 ms to 31.21 ± 7.2 ms, which is a difference of −5.67 ± 2.2 ms (p = 0.031)), and at 12 months in the superficial layer of the lesion in patients at 3 T (decrease from 41.89 ± 9.3 ms to 35.28 ± 4.9 ms, which is a difference of −6.60 ± 4.4 ms (p = 0.044). No significant differences were recorded at 7 T. CONCLUSION: The change in T(2) values acquired with 3 T 3D-TESS appears to be reflecting subtle changes of cartilage composition in the course of low-grade lesion development. 7 T T(2) mapping does not reflect these changes probably due to completely decayed short T(2) component.

We investigated whether nasal chondrocytes (NC) can be used to generate composite constructs with properties necessary for the repair of osteochondral (OC) lesions, namely maturation, integration and capacity to recover from inflammatory burst. OC grafts were fabricated by combining engineered cartilage tissues (generated by culturing NC or articular chondrocytes - AC - onto Chondro-Gide® matrices) with devitalized spongiosa cylinders (Tutobone®). OC tissues were then exposed to IL-1β for three days and cultured for additional 2 weeks in the absence of IL-1β. Cartilage maturation extent was assessed (immune) histologically, biochemically and by delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) while cartilage/bone integration was assessed using a peel-off mechanical test. The use of NC as compared to AC allowed for more efficient cartilage matrix accumulation and superior integration of the cartilage/bone layers. dGEMRIC and biochemical analyzes of the OC constructs showed a reduced glycosaminoglycan (GAG) contents upon IL-1β administration. Cartilaginous matrix contents and integration forces returned to baseline up on withdrawal of IL-1β. By having a cartilage layer well developed and strongly integrated to the subchondral layer, OC tissues generated with NC may successfully engraft in an inflammatory post-surgery joint environment.

Despite the significant progress in the field of bone tissue engineering, cell-based products have not yet reached the stage of clinical adoption. This is due to the uncertain advantages from the standard-of-care, combined with challenging cost-and regulatory-related issues. Novel therapeutic approaches could be based on exploitation of the intrinsic regenerative capacity of bone tissue, provided the development of a deeper understanding of its healing mechanisms. While it is well-established that endogenous progenitors can be activated toward bone formation by overdoses of single morphogens, the challenge to stimulate the healing processes by coordinated and controlled stimulation of specific cell populations remains open. Here, we review the recent approaches to generate osteoinductive materials based on the use of decellularized extracellular matrices (ECM) as reservoirs of multiple factors presented at physiological doses and through the appropriate ligands. We then propose the generation of customized engineered and decellularized ECM (i) as a tool to better understand the processes of bone regeneration and (ii) as safe and effective "off-the-shelf" bone grafts for clinical use. This article is part of a Special Issue entitled Stem Cells and Bone.

Meniscus regeneration is an unsolved clinical challenge. Despite the wide acceptance of the degenerative consequences of meniscectomy, no surgical procedure has succeeded to date in regenerating a functional and long-lasting meniscal fibrocartilage. Research proposed a number of experimental approaches encompassing all the typical strategies of regenerative medicine: cell-free scaffolds, gene therapy, intra-articular delivery of progenitor cells, biological glues for enhanced bonding of reparable tears, partial and total tissue engineered meniscus replacement. None of these approaches has been completely successful and can be considered suitable for all patients, as meniscal tears require specific and patient-related treatments depending on the size and type of lesion. Recent advances in cell biology, biomaterial science and bioengineering (e.g., bioreactors) have now the potential to drive meniscus regeneration into a series of clinically relevant strategies. In this tutorial paper, the clinical need for meniscus regeneration strategies will be explained, and past and current experimental studies on meniscus regeneration will be reported.

Standard cartilage tissue engineering approaches, for example, matrix-induced autologous chondrocyte implantation (MACI), consist of the implantation of cell-based constructs whose survival and further development first depend on the degree of graft maturity at the time of surgery (e.g., matrix production) and, subsequently, on initial host reaction. Indeed, blood vessel ingrowth and macrophage migration within the implant may endanger graft stability of immature constructs; so, control of angiogenesis was proposed as an adjuvant of cellular therapy for the treatment of cartilage defects. In this study, we hypothesized that engineered constructs with no in vitro precultivation, but functionalized to block angiogenesis right on implantation, might result in better survival, as well as superior long-term cartilaginous quality. Here, we propose a clinically compatible fibrin/hyaluronan scaffold seeded with nasal chondrocytes (NC) and functionalized with an FDA-approved anti-angiogenic drug (bevacizumab; Avastin(®)), which sequestrates vascular endothelial growth factor from the surrounding environment. Our results show that the sustained bevacizumab release from NC-loaded scaffolds after subcutaneous implantation in nude mice efficiently blocked host vessels ingrowth (five times lower CD31(+) cells infiltration vs. control group, at 3 weeks after implant), and enhanced constructs survival rate (75% vs. 18% for the control, at 6 weeks after implant). In vitro assays, developed to elucidate the role of specific construct components in the in vivo remodeling, allowed to determine that fibrin degradation products enhanced the in vitro endothelial cell proliferation, as well as the macrophage migration; whereas the presence of bevacizumab was capable of counteracting these effects. The proposed pharmacological control of angiogenesis by a therapeutic drug released from a scaffold might enhance cartilage regeneration by MACI approaches, possibly allowing it to bypass the complex and costly phase of graft preculture to gain increased functionality.

The meniscus plays an important role in knee function and mechanics. Meniscal lesions, however, are common phenomena and this tissue is not able to achieve spontaneous successful repair, particularly in the inner avascular zone. Several animal models have been studied and proposed for testing different reparative approaches, as well as for studying regenerative methods aiming to restore the original shape and function of this structure. This review summarizes the gross anatomy, function, ultrastructure and biochemical composition of the knee meniscus in several animal models in comparison with the human meniscus. The relevance of the models is discussed from the point of view of basic research as well as of clinical translation for meniscal repair, substitution and regeneration. Finally, the advantages and disadvantages of each model for various research directions are critically discussed.

Bone development, growth, and repair predominantly occur through the process of endochondral ossification, characterized by remodelling of cartilaginous templates. The same route efficiently supports engineering of bone marrow as a niche for hematopoietic stem cells (HSC). Here we describe a combined in vitro/in vivo system based on bone marrow-derived Mesenchymal Stem/Stromal Cells (MSC) that duplicates the hallmark cellular and molecular events of endochondral ossification during development. The model requires MSC culture with instructive molecules to generate hypertrophic cartilage tissues. The resulting constructs complete the endochondral route upon in vivo implantation, in the timeframe of up to 12 weeks. The described protocol is clearly distinct from the direct ossification approach typically used to drive MSC towards osteogenesis. Recapitulation of endochondral ossification allows modelling of stromal-HSC interactions in physiology and pathology and allows engineering processes underlying bone regeneration.

Background:Rotator cuff repair typically results in a satisfactory, although variable, clinical outcome. However, anatomic failure of the repaired tendon often occurs.Hypothesis:Patch augmentation can improve the results of open rotator cuff repair by supporting the healing process, protecting the suture, and reducing friction in the subacromial space.Study Design:Cohort study; Level of evidence, 3.Methods:A total of 152 patients with a posterosuperior massive rotator cuff tear were treated by open repair only (control group; n = 51; mean age, 67.06 ± 4.42 years), open repair together with collagen patch augmentation (collagen group; n = 49; mean age, 66.53 ± 5.17 years), or open repair together with polypropylene patch augmentation (polypropylene group; n = 52; mean age, 66.17 ± 5.44 years) and were retrospectively studied. Patients were evaluated preoperatively and after 36 months with a visual analog scale (VAS) and the University of California, Los Angeles (UCLA) shoulder rating scale and by measuring elevation of the scapular plane and strength with a dynamometer. The VAS and UCLA scores were also obtained 2 months postoperatively. Tendon integrity was assessed after 1 year by ultrasound. Patients were homogeneous as per the preoperative assessment.Results:After 2 months, results (mean ± standard deviation) for the control, collagen, and polypropylene groups, respectively, were as follows: VAS scores were 6.96 ± 1.11, 6.46 ± 1.02, and 4.92 ± 0.90, while UCLA scores were 11.29 ± 1.46, 11.40 ± 1.51, and 19.15 ± 1.99. After 36 months, the mean scores for the respective groups were 3.66 ± 1.05, 4.06 ± 1.02, and 3.28 ± 1.10 for the VAS and 14.88 ± 1.98, 14.69 ± 1.99, and 24.61 ± 3.22 for the UCLA scale. In addition, after 36 months, elevation on the scapular plane was 140.68° ± 9.84°, 140.61° ± 12.48°, and 174.71° ± 8.18°, and abduction strength was 8.73 ± 0.54 kg, 9.03 ± 0.60 kg, and 13.79 ± 0.64 kg for the control, collagen, and polypropylene groups, respectively. The retear rate after 12 months was 41% (21/51) for the control group, 51% (25/49) for the collagen group, and 17% (9/52) for the polypropylene group. In particular, the reduced 12-month retear rate and the increased UCLA scores, abduction strength, and elevation at 3-year follow-up were statistically significant for patients treated with a polypropylene patch compared with those treated with repair only or with a collagen patch.Conclusion:Polypropylene patch augmentation of rotator cuff repair was demonstrated to significantly improve the 36-month outcome in terms of function, strength, and retear rate.

Osteochondral lesions of the talus can occur after ankle trauma (e.g., inversion sprains) or be idiopathic, in case of osteochondritis dissecans. In both scenarios, it is challenging for the orthopedic surgeon to establish the optimal therapy. Treatment options include both conservative and surgical procedures depending on severity, size, and location of the lesion. The aim of surgery is to restore the articular surface and, therefore, possibly avoid or delay the onset of osteoarthritis. Thanks to the recent advancements in tissue engineering and regenerative medicine, scaffold-based techniques, either cell-free or cell based, have been proposed for cartilage repair in the ankle. The optimal scaffold should simultaneously promote high rates of cell division and cartilaginous extracellular matrix synthesis by phenotypically stable chondrocytes. In this chapter, general characteristics of scaffolds and the main scaffolds and procedures used for cartilage repair in the ankle will be discussed.

Currently, autologous bone grafting represents the clinical gold standard in orthopaedic surgery. In certain cases, however, alternative techniques are required. The clinical utility of stem and stromal cells has been demonstrated for the repair and regeneration of craniomaxillofacial and long bone defects although clinical adoption of bone tissue engineering protocols has been very limited. Initial tissue engineering studies focused on the bone marrow as a source of cells for bone regeneration, and while a number of promising results continue to emerge, limitations to this technique have prompted the exploration of alternative cell sources, including adipose and muscle tissue. In this review paper we discuss the advantages and disadvantages of cell sources with a focus on adipose tissue and the bone marrow. Additionally, we highlight the relatively recent paradigm of developmental engineering, which promotes the recapitulation of naturally occurring developmental processes to allow the implant to optimally respond to endogenous cues. Finally we examine efforts to apply lessons from studies into different cell sources and developmental approaches to stimulate bone growth by use of decellularised hypertrophic cartilage templates.

Bone development, growth, and repair predominantly occur through the process of endochondral ossification, characterized by remodelling of cartilaginous templates. The same route efficiently supports engineering of bone marrow as a niche for hematopoietic stem cells (HSC). Here we describe a combined in vitro/in vivo system based on bone marrow-derived Mesenchymal Stem/Stromal Cells (MSC) that duplicates the hallmark cellular and molecular events of endochondral ossification during development. The model requires MSC culture with instructive molecules to generate hypertrophic cartilage tissues. The resulting constructs complete the endochondral route upon in vivo implantation, in the timeframe of up to 12 weeks. The described protocol is clearly distinct from the direct ossification approach typically used to drive MSC towards osteogenesis. Recapitulation of endochondral ossification allows modelling of stromal-HSC interactions in physiology and pathology and allows engineering processes underlying bone regeneration.

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

Posterior spinal instrumentation is frequently used for the treatment of spine disorders. Importantly, different requirements have to be considered for the optimal use of these systems in various clinical scenarios. In this work, we focused on the role of rods diameter on hardware's stiffness. For this purpose, we established an in vitro model and compared the response to axial load of a posterior stabilization system, characterized by rods of different diameter (4, 5, 6 mm), with that of Dynesys®. Intuitively, the higher the stiffness of the hardware, the lower the load is transferred to the disc. However, the 4 hardware tested showed a different trend in the response to the load regimens: when increasing the load, more flexible systems display a progressive reduction in the percentage of load which is transferred to the disc while more rigid system display the opposite trend. Considering that the load which is transferred, and not by-passed by the hardware, influences the healing of a fracture; the integration of a bone graft or a cage; the fusion process, these data have a relevant impact on clinical practice and highlight features that have to be considered in the choice for the optimal posterior spinal instrumentation.

Objective: The purpose of our study was to determine the effectiveness of cartilage repair utilizing 1-step surgery with bone marrow aspirate concentrate (BMAC) and a collagen I/III matrix (Chondro-Gide, Geistlich, Wolhusen, Switzerland). Materials and Methods: We prospectively followed up for 2 years 15 patients (mean age, 48 years) who were operated for grade IV cartilage lesions of the knee. Six of the patients had multiple chondral lesions; the average size of the lesions was 9.2 cm2. All patients underwent a mini-arthrotomy and concomitant transplantation with BMAC covered with the collagen matrix. Coexisting pathologies were treated before or during the same surgery. X-rays and MRI were collected preoperatively and at 1 and 2 years’ follow-up. Visual analog scale (VAS), International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm, Marx, SF-36 (physical/mental), and Tegner scores were collected preoperatively and at 6, 12, and 24 months’ follow-up. Four patients gave their consent for second-look arthroscopy and 3 of them for a concomitant biopsy. Results: Patients showed significant improvement in all scores at final follow-up ( P < 0.005). Patients presenting single lesions and patients with small lesions showed higher improvement. MRI showed coverage of the lesion with hyaline-like tissue in all patients in accordance with clinical results. Hyaline-like histological findings were also reported for all the specimens analyzed. No adverse reactions or postoperative complications were noted. Conclusion: This study showed that 1-step surgery with BMAC and collagen I/III matrix could be a viable technique in the treatment of grade IV knee chondral lesions.

The aim of this study was to investigate the in vitro effect of different concentrations of blood on the morphological and biochemical properties of engineered cartilage. Previous studies have demonstrated a negative effect of blood on native cartilage; however, the effect of the contact of blood on engineered cartilage is unclear. Articular chondrocytes were isolated from swine joints, expanded in monolayer culture, and seeded onto collagen membranes. The seeded membranes were cultured for 3 days in the presence of different concentrations of peripheral blood. Some samples were retrieved at the end of the blood contact, others after 21 additional days of standard culture conditions, in order to investigate the “long-term effect” of the blood contact. All seeded samples showed an increase in the weight and an evident cartilage-like matrix production. A concentration-dependent reduction in the mitochondrial activity due to blood contact was shown at the earlier culture time, followed by a partial recover at the longer culture time. A blood contact of 3 days affected the chondrocytes’ activity and determined a delay in the maturation of the engineered cartilage. These findings have clinical relevance, as autologous chondrocytes seeded onto biological scaffolds has become an established surgical method for articular cartilage repair. Therefore, further investigation into material sciences should be encouraged for the development of scaffold protecting the reparative cells from the blood insult.

Menisci represent fundamental structures for the maintenance of knee homeostasis, playing a key role in knee biomechanics. However, their intrinsic regenerative potential is poor. As a consequence, when a lesion occurs and the meniscus is partially removed by surgery, knee mechanics is subject to dramatic changes. These have been demonstrated to lead often to the development of early osteoarthritis. Therefore, menisci should be repaired whenever possible. In the last decades, tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of an articular chondrocytes-fibrin glue hydrogel was tested as a biologic glue to improve the bonding between two swine meniscal slices in a nude mouse model. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for 4 weeks. Upon retrieval, a firm gross bonding was observed in the experimental samples while none of the control samples, prepared with acellular fibrin glue at the interface, presented any sign of bonding. This was consistent with the histological and scanning electron microscope findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, partially penetrating the native meniscus tissue. In order to overcome the lack of regenerative properties of the meniscus, the rationale of using cellular fibrin glue is that fibrin provides immediate stability while carrying cells in the site of lesion. Moreover, fibrin gel is recognized as an optimal scaffold for cell embedding and for promoting fibrocartilaginous differentiation of the cells which synthesize matrix having healing property. These results demonstrated the potential of this model for improving the meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its potential for clinical application.

Meniscus repair is a current clinical challenge. Menisci play a fundamental role in knee biomechanics, but they lack intrinsic regenerative properties. Conse quently, when a tear occurs and the meniscus is removed surgically, even partially, crucial changes in knee homeostasis take place, often leading to the development of early osteoarthritis. In recent decades tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of isolated chondrocytes was analysed in a nude mouse meniscus repair model: a swine chondrocyte-fibrin glue suspension was utilised as a biologic glue to improve bonding between two meniscal slices obtained from swine menisci. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for four weeks. Upon retrieval, a firm gross bonding was observed. This was consistent with the histological findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, having some penetration buds arising from the neo-tissue. These results demonstrated the potential of this model for improving meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its feasibility in clinical practice.

Fracture of the humerus represents a common problem among the young and elderly populations. Although humerus fractures usually heal uneventfully, nonunions can sometimes occur. We present a case of humeral nonunion managed with shock wave therapy in the outpatient setting. A 62-year-old woman with a closed comminuted fracture of the proximal third of the humerus came to our attention 6 months after the trauma with a hypertrophic nonunion. Radiographs showed a hypertrophic callus with a fracture gap of 4 mm. We performed ten shock wave treatments in the outpatient department, with an interval of 60 days between each single treatment. Each session consisted of 2000 impulses at 0.86 mJ/mm2 applied in two planes. No anesthesia was given during the treatment. The patient received a sling to support the treated arm during the first days following each treatment. Follow-up assessment by radiographic and physical examination, performed after 4 weeks and at 3, 6, 12, 18 and 24 months, showed complete bony union and cortical bridging, achieved at the end of the treatment. We believe that this method is a safe and effective alternative to surgery for the treatment of chronic hypertrophic nonunions. Moreover, in case such treatment is unsuccessful, subsequent surgery is not precluded.

The role of fresh allogeneic osteochondral allograft transplantation (OCA) in the cartilage repair algorithm has been long debated and this procedure is primarily considered as a salvage procedure, to be used when other, simple, techniques have failed. Gracitelli et al. in a retrospective comparison of patients who received OCA as primary treatment or as a salvage procedure, demonstrates that the outcome of this procedure is minimally influenced by a previous failed treatment and that OCA represents an effective solution for both primary and revision surgery of chondral and osteochondral lesions of the knee. In particular, optimal indications for OCA seem to be revision of previously failed bone marrow stimulation techniques with an impaired subchondral bone plate and primary treatment of large osteochondral defects.

The aim of this study is to prospectively evaluate the medium-term effectiveness and regenerative capability of autologous adult mesenchymal stem cells, harvested as bone marrow aspirate concentrate (BMAC), along with a hyaluronan-based scaffold (Hyalofast) in the treatment of ICRS grade 4 chondral lesions of the knee joint, in patients older than 45 years.A study group of 20 patients with an age45 years (mean 50.0 ± 4.1 years) was compared to a control group of 20 patients with an age45 years (mean 36.6 ± 5.0). Patients were prospectively evaluated for 4 years. All patients were evaluated with MRI, KOOS, IKDC, VAS and Tegner scores preoperatively and at two-year and final follow-up.At final follow-up, all scores significantly improved (P 0.001) as follows: all KOOS score categories; Tegner 2 (range 0-4) to 6 (range 4-8) and 3 (range 0-6) to 6 (range 3-10); IKDC subjective (39.2 ± 16.5 to 82.2 ± 8.9) and (40.8 ± 13.9 to 79.4 ± 14.6), in the study and control group respectively. In addition, we show that results are affected by lesion size and number but not from concomitant surgical procedures. MRI showed complete filling in 80 % of patients in the study group and 71 % of patients in the control group. Histological analysis conducted in three patients from the study and two patients from the control group revealed good tissue repair with a variable amount of hyaline-like tissue.Treatment of cartilage lesions with BMAC and Hyalofast is a viable and effective option that is mainly affected by lesion size and number and not by age. In particular, it allows to address the45 years population with functional outcomes that are comparable to younger patients at final follow-up.Prospective cohort study, Level II.

We investigated whether nasal chondrocytes (NC) can be used to generate composite constructs with properties necessary for the repair of osteochondral (OC) lesions, namely maturation, integration and capacity to recover from inflammatory burst. OC grafts were fabricated by combining engineered cartilage tissues (generated by culturing NC or articular chondrocytes - AC - onto Chondro-Gide® matrices) with devitalized spongiosa cylinders (Tutobone®). OC tissues were then exposed to IL-1β for three days and cultured for additional 2 weeks in the absence of IL-1β. Cartilage maturation extent was assessed (immune) histologically, biochemically and by delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) while cartilage/bone integration was assessed using a peel-off mechanical test. The use of NC as compared to AC allowed for more efficient cartilage matrix accumulation and superior integration of the cartilage/bone layers. dGEMRIC and biochemical analyzes of the OC constructs showed a reduced glycosaminoglycan (GAG) contents upon IL-1β administration. Cartilaginous matrix contents and integration forces returned to baseline up on withdrawal of IL-1β. By having a cartilage layer well developed and strongly integrated to the subchondral layer, OC tissues generated with NC may successfully engraft in an inflammatory post-surgery joint environment.

In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be dis- tinguished from mesoderm-derived cells by their Hox-negative profile—a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differen- tiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autol- ogous NC-based engineered tissues for the treatment of traumatic articular cartilage lesions.

Although relatively uncommon, spontaneous healing from a meniscus injury has been observed even within the avascular area. This may be the result of the existence of mesenchymal stem cells in synovial fluid.The purpose of this study was to investigate whether mesenchymal stem cells existed in the synovial fluid of the knee after meniscus injury.Synovial fluid was obtained from the knees of 22 patients with meniscus injury just before meniscus surgery and from 8 volunteers who had no history of knee injury. The cellular fraction of the synovial fluid was cultured for 14 days followed by analysis for multilineage potential and presentation of surface antigens characteristic of mesenchymal stem cells. Colony-forming efficiency and proliferation potential were also compared between the two groups.Cells with characteristics of mesenchymal stem cells were observed in the synovial fluid of injured knees to a much greater degree than in uninjured knees. The colony-forming cells derived from the synovial fluid of the knee with meniscus injury had multipotentiality and surface epitopes identical to mesenchymal stem cells. The average number of colony formation, obtained from 1 mL of synovial fluid, in meniscus-injured knees was 250, higher than that from healthy volunteers, which was 0.5 (p 0.001). Total colony number per synovial fluid volume was positively correlated with the postinjury period (r = 0.77, p 0.001).Mesenchymal stem cells were found to exist in synovial fluid from knees after meniscus injury. Mesenchymal stem cells were present in higher numbers in synovial fluid with meniscus injury than in normal knees. Total colony number per synovial fluid volume was positively correlated with the postinjury period.Our current human study and previous animal studies suggest the possibility that mesenchymal stem cells in synovial fluid increase after meniscus injury contributing to spontaneous meniscus healing.

Embryonic development, lengthening, and repair of most bones proceed by endochondral ossification, namely through formation of a cartilage intermediate. It was previously demonstrated that adult human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) can execute an endochondral program and ectopically generate mature bone. Here we hypothesized that hMSCs pushed through endochondral ossification can engineer a scaled-up ossicle with features of a “bone organ,” including physiologically remodeled bone, mature vasculature, and a fully functional hematopoietic compartment. Engineered hypertrophic cartilage required IL-1β to be efficiently remodeled into bone and bone marrow upon subcutaneous implantation. This model allowed distinguishing, by analogy with bone development and repair, an outer, cortical-like perichondral bone, generated mainly by host cells and laid over a premineralized area, and an inner, trabecular-like, endochondral bone, generated mainly by the human cells and formed over the cartilaginous template. Hypertrophic cartilage remodeling was paralleled by ingrowth of blood vessels, displaying sinusoid-like structures and stabilized by pericytic cells. Marrow cavities of the ossicles contained phenotypically defined hematopoietic stem cells and progenitor cells at similar frequencies as native bones, and marrow from ossicles reconstituted multilineage long-term hematopoiesis in lethally irradiated mice. This study, by invoking a “developmental engineering” paradigm, reports the generation by appropriately instructed hMSC of an ectopic “bone organ” with a size, structure, and functionality comparable to native bones. The work thus provides a model useful for fundamental and translational studies of bone morphogenesis and regeneration, as well as for the controlled manipulation of hematopoietic stem cell niches in physiology and pathology.

The meniscus plays an important role in knee function and mechanics. Meniscal lesions, however, are common phenomena and this tissue is not able to achieve spontaneous successful repair, particularly in the inner avascular zone. Several animal models have been studied and proposed for testing different reparative approaches, as well as for studying regenerative methods aiming to restore the original shape and function of this structure. This review summarizes the gross anatomy, function, ultrastructure and biochemical composition of the knee meniscus in several animal models in comparison with the human meniscus. The relevance of the models is discussed from the point of view of basic research as well as of clinical translation for meniscal repair, substitution and regeneration. Finally, the advantages and disadvantages of each model for various research directions are critically discussed.

Previous studies showed that human nasal chondrocytes (HNC) exhibit higher proliferation and chondrogenic capacity as compared to human articular chondrocytes (HAC). To consider HNC as a relevant alternative cell source for the repair of articular cartilage defects it is necessary to test how these cells react when exposed to environmental factors typical of an injured joint. We thus aimed this study at investigating the responses of HNC and HAC to exposure to interleukin (IL)-1? and low oxygen. For this purpose HAC and HNC harvested from the same donors (N=5) were expanded in vitro and then cultured in pellets or collagen-based scaffolds at standard (19%) or low oxygen (5%) conditions. Resulting tissues were analyzed after a short (3 days) exposure to IL-1?, mimicking the initially inflammatory implantation site, or following a recovery time (1 or 2 weeks for pellets and scaffolds, respectively). After IL-1? treatment, constructs generated by both HAC and HNC displayed a transient loss of GAG (up to 21.8% and 36.8%, respectively) and, consistently, an increased production of metalloproteases (MMP)-1 and -13. Collagen type II and the cryptic fragment of aggrecan (DIPEN), both evaluated immunohistochemically, displayed a trend consistent with GAG and MMPs production. HNC-based constructs exhibited a more efficient recovery upon IL-1? withdrawal, resulting in a higher accumulation of GAG (up to 2.6-fold) compared to the corresponding HAC-based tissues. On the other hand, HAC displayed a positive response to low oxygen culture, while HNC were only slightly affected by oxygen percentage. Collectively, under the conditions tested mimicking the postsurgery articular environment, HNC retained a tissue-forming capacity, similar or even better than HAC. These results represent a step forward in validating HNC as a cell source for cartilage tissue engineering strategies.

Despite the compelling clinical needs in enhancing bone regeneration and the potential offered by the field of tissue engineering, the adoption of cell-based bone graft substitutes in clinical practice is limited to date. In fact, no study has yet convincingly demonstrated reproducible clinical performance of tissue-engineered implants and at least equivalent cost-effectiveness compared to the current treatment standards. Here, we propose and discuss how tissue engineering strategies could be evolved towards more efficient solutions, depicting three different experimental paradigms: (i) bioreactor-based production; (ii) intraoperative manufacturing, and (iii) developmental engineering. The described approaches reflect the need to streamline graft manufacturing processes while maintaining the potency of osteoprogenitors and recapitulating the sequence of biological steps occurring during bone development, including vascularization. The need to combine the assessment of efficacy of the different strategies with the understanding of their mechanisms of action in the target regenerative processes is highlighted. This will be crucial to identify the necessary and sufficient set of signals that need to be delivered at the injury or defect site and should thus form the basis to define release criteria for reproducibly effective engineered bone graft substitutes.

"Thoracic outlet syndrome is an often misdiagnosed syndrome which consists of a neurovascular compression at the upper thoracic outlet. The clinical presentation can be variable, ranging from mild symptoms to venous thrombosis and muscle atrophy. Many aetiologies, both congenital and acquired, related either to bony or soft tissue anomalies, have been associated with this syndrome. As a consequence, the diagnosis is often challenging and sometimes it can be obtained only with surgical exploration. Additionally, no specific clinical test is considered diagnostic of thoracic outlet syndrome. However, the recent advances in imaging techniques together with a careful clinical evaluation give the surgeon the chance to recognize the constricting anatomy before surgery in many cases. No standard surgical procedure has been identified; however, in literature the largest series have been treated with transaxillary first rib resection. Here we report our experience in the surgical treatment of this syndrome with a minimum follow-up of three years. Our approach consists of performing a supraclavicular decompression without routine first rib resection. This allows for identifying and removing the constricting anatomy in most cases, with satisfactory results in 96.9% of patients and a low complication rate."

In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone) using a fibrin gel (Tisseel). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and--since it is based on clinically compliant cells and materials--is amenable to be readily tested in the clinic.

Research in tissue engineering has been focused on articular cartilage repair for more than a decade. Some pioneristic studies involved the use of hydrogels such as alginate and fibrin glue which still possess valuable potential for cartilage regeneration. One of the main issues in cartilage tissue engineering is represented by the ideal maturation of the construct, before in vivo implantation, in order to optimize matrix quality and integration. The present study was focused on the effect of in vitro culture on a fibrin glue hydrogel embedding swine chondrocytes. We performed an evaluation of the immunohistochemical and biochemical composition and of the biomechanical properties of the construct after 1 and 5 weeks of culture. We noticed that chondrocytes survived in the fibrin glue gel and enhanced their synthetic activity. In fact, DNA content remained stable, while all indices of cartilage matrix production increased (GAGs content, immunohistochemistry for collagen II and safranin-o staining). On the other hand, the biomechanical properties remained steady, indicating a gradual substitution of the hydrogel scaffold by cartilaginous matrix. This demonstrates that an optimal preculture could provide the surgeon with a better engineered cartilage for implantation. However, whether this more mature tissue will result in a more efficient regeneration of the articular surface still has to be evaluated in future investigations.

Articular cartilage lesions have a poor intrinsic healing potential. The repair tissue is often fibrous, having insufficient biomechanical properties, which could frequently lead to the development of early osteoarthritis. In the last decade, tissue engineering approaches addressed this topic in order to restore joint function with a differentiated and functional tissue. Many biomaterials and techniques have been proposed and some of them applied in clinical practice, even though several concerns have been raised on the quality of the engineered tissue and on its integration in the host joint. In this study, we focused on engineering in vitro a biphasic composite made of cellular fibrin glue and a calcium-phosphate scaffold. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to allow a more efficient integration of the engineered tissue with the host. However, a firm in vitro bonding between the two components of the composite is a necessary condition to validate this model. Our study demonstrated a gross and microscopic integration of the two components and a cartilage-like quality of the newly formed matrix. Moreover, we noticed an improvement of this integration and GAGs production during the in vitro culture.

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days' contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the "long-term effect" of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.

Pseuodaneurysms are an extremely rare complication of osteochondromas. We describe a case of traumatic pseudoaneurysm of the brachial artery presenting as a soft tissue mass in a patient who was treated for an osteochondroma 3 years earlier. This case demonstrates that radiographic follow-up of large osteochondromas is mandatory and that, in patients with soft tissue masses and a history of osteochondroma, pseudoaneurysms should be included in the differential diagnosis.

BackgroundThe humerus is the second most common site of metastatic bone disease involving long bones. Tumors which have a predilection for dissemination to bone are those of breast, prostate, thyroid, lung and kidney. The rationale for surgical treatment of these lesions is to prevent or treat pathological fractures in order to relieve pain and improve function.Materials and methodsForty patients who had resection of the proximal humerus for metastatic bone disease and reconstruction with a modular prosthesis were retrospectively reviewed.ResultsMean functional outcome was 73.1% (Enneking score) and better results were achieved when a reverse prosthesis was implanted. Overall survival was 70% at 1 year, 42.5% at 2 years and 20% at 5 years. Local recurrence occurred in 4 patients, each of whom had initially been treated for a pathological fracture.ConclusionsIt is important to follow rational guidelines, like those of Capanna and Mirels, in order to prevent pathological fractures and to give the patient a definitive treatment, as the advances in the management of cancer prolong the survival of these patients. In this series, satisfactory results were obtained, giving the patients an acceptable quality of life.