Force-feeding malignant mesothelioma stem-cell like with exosome-delivered miR-126 induces tumour cell killing.

Malignant pleural mesothelioma (MPM) is an aggressive tumour resistant to treatments. It has been postulated that cancer stem cells (CSCs) persist in tumours causing relapse after multimodality treatment. In the present study, a novel miRNA-based therapy approach is proposed. MPM-derived spheroids have been treated with exosome-delivered miR-126 (exo-miR) and evaluated for their anticancer effect. The exo-miR treatment increased MPM stem-cell like stemness and inhibited cell proliferation. However, at a prolonged time, the up taken miR-126 was released by the cells themselves through exosomes; the inhibition of exosome release by an exosome release inhibitor GW4869 induced miR-126 intracellular accumulation leading to massive cell death and in vivo tumour growth arrest. Autophagy is involved in these processes; miR-126 accumulation induced a protective autophagy and the inhibition of this process by GW4869 generates a metabolic crisis that promotes necroptosis, which was associated with PARP-1 over-expression and cyt-c and AIF release. Here, for the first time, we proposed a therapy against CSCs, a heterogeneous cell population involved in cancer development and relapse.

Assessing 3-D Printing in Hip Replacement Surgical Planning.

PURPOSE: To evaluate the accuracy of 3-D printed models of the femoral head based on preoperative computed tomography (CT) images. Other goals were to compare the cartilage thickness of bony specimen to the printed models and calculate the standard deviation between 3-D printed models based on CT images and laser scan models. METHODS: This retrospective study analyzed 10 patients who underwent preoperative CT imaging and hip replacement. Preoperative femoral head 3-D printed models were produced from CT images. Bony specimens were collected from surgical operations and scanned using CT and 3-D laser scanning, and cartilage thickness subsequently was measured by histological analysis. Comparisons of printed models based on CT images and printed models based on 3-D laser scanning were performed by overlapping their external surfaces using dedicated software and the standard deviation was calculated. RESULTS: The average standard deviation between the bony specimen 3-D models and preoperative 3-D printed CT femoral head models was 0.651 mm. The cartilage was approximately 1.487 mm thick. DISCUSSION: The comparison between preoperative CT image-based 3-D models and the postoperative bony specimen-based models permitted evaluation of the accuracy of preoperative CT image-based 3-D printed models. Cartilage thickness was estimated indirectly by comparing models obtained by CT and laser scanning, and it was related to the calculated standard deviation to overcome the cartilage detection limit of CT. This study shows how each step can generate accuracy errors on the final 3-D printed model. A repeatable and sustainable workflow for creating accurate and reproducible 3-D printed models could overcome this issue. Moreover, orthopedic surgeons should be aware of 3-D printed model precision in clinical practice. CONCLUSIONS: This study provides encouraging results on the accuracy of 3-D printed models for surgical planning.

Suture anchor reinsertions of distal biceps rupture: a histologic analysis of a torn tendon and clinical results at short- and long-term follow-up.

BACKGROUND: Distal biceps brachii tendon (DBBT) rupture is a relatively rare injury. Nonsurgical treatment determines 30%-40% power loss of elbow flexion and up to 50% of forearm supination. Therefore, refixation of the DBBT is recommended. The DBBT is exposed to tension and compression loading. It is known that the tendon under compression might develop fibrocartilaginous metaplasia that improves the resistance to compression but reduces the resistance to tension. To test this hypothesis, the present study evaluated the presence of cartilage in DBBT samples. Furthermore, the present study evaluated the clinical and functional outcomes of anatomic reinsertion through suture anchors in a cohort of patients after 1, 3, and 5 years of follow-up. METHODS: Between 2011 and 2014, 21 patients with DBBT tear underwent a suture anchor reattachment. Histochemical and immunohistochemical analysis of torn samples of DBBT collected at the time of surgical repair were performed to test the presence of cartilage. During the follow-up examination, mobility, elbow radiographs, Mayo Elbow Performance Score, and isokinetic analysis were prospectively evaluated. RESULTS: Fibrocartilage was detected in all tendon samples collected. Two cases of transient paresthesia in the lateral antebrachial cutaneous nerve occurred, but they resolved in 6 weeks. There were no vascular deficits, re-ruptures, radioulnar synostoses, or infective complications at follow-up. Three patients reported loss of supination. Mayo Elbow Performance Score showed good and excellent clinical and functional results. No significant differences about strength and fatigue in flexion-supination were recorded between the surgical and contralateral side at 3 and 5 years of follow-up. Arm dominance influenced supination but not flexion. CONCLUSION: On the basis of our results, we find that the presence of cartilage metaplasia might make the DBBT at higher risk of rupture assuming the compression loading and the hypovascular zone of the tendon. However, concerning the lack of histologic analysis of the healthy DBBT, its role in tendon pathology remains to be clearly defined. The technique of suture anchor reinsertion by a single incision was shown to be safe, with few complications and good functional results at 5 years of follow-up. No significant differences were reported between the injured and noninjured side in terms of flexion and supination isokinetic analysis, whereas arm dominance had a positive effect on supination.

Mechanical and Enzymatic Procedures to Isolate the Stromal Vascular Fraction From Adipose Tissue: Preliminary Results.

Adipose-derived MSCs (ASCs) and stromal vascular fraction (SVF) play an important role in regenerative medicine and in the treatment of osteoarthritis. ASCs extracted from lipoaspirates are a valuable cell source due to their abundance and accessibility. ASCs are retrieved from the aqueous fraction of the digested lipoaspirate. The aqueous fraction is known as SVF and includes, ASCs, endothelial precursor cells (EPCs), endothelial cells (ECs), macrophages, smooth muscle cells, lymphocytes, pericytes, as well as pre-adipocytes. To date, two types of techniques to isolate SVF have been proposed: enzymatic and mechanical. The enzymatic method is particularly indicated in SVF isolation since it disrupts the extracellular matrix (ECM) and the binding of adipocytes and other cells but is restricted by regulatory issues related to enzymatic procedures, especially within the European Community. Thus, making the search for alternative mechanical methods imperative. This study assesses the SVF harvested from subcutaneous abdominal fat via two different mechanical procedures and the standard enzymatic method to evaluate their eligibility in a clinical context. In particular, we analyze cell viability (at 0 and after 72 h) as well as the expression of cluster differentiation (CD) for each sample and the differentiation in adipocytic, chondrocytic, osteocytic linage. The mechanical procedures yielded no significant difference in cell viability and cluster differentiation pattern expression, even if enzymatic procedure still remain the "gold standard." We retain that clinical efficacy in treating ostheoarthrosis with SVF administration is probably related to his anti-inflammatory and immunoregulatory effect, rather than the ability to differentiate in specific cell lineage. However, further studies are required to support and improve our findings.

Detecting senescent fate in mesenchymal stem cells: a combined cytofluorimetric and ultrastructural approach.

Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover-like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life-adds a new piece to the regenerative medicine jigsaw in an ageing society.

Fibrocartilaginous metaplasia identified in the long head of the biceps brachii.

BACKGROUND: In the glenohumeral joint, the long head of biceps brachii (LHBB) is exposed to tension and compression loading. The short head of biceps brachii (SHBB) works only in tension. It is known that tendon under compression might develop fibrocartilaginous metaplasia that improves the resistance to compression but reduces the resistance to tension. This study evaluated the presence of cartilage in LHBB and SHBB samples, supporting its possible role in tendon tear. METHODS: Between 2014 and 2016, 13 samples of LHBB and SHBB were collected during surgery for shoulder instability, glenohumeral arthritis, and massive rotator cuff tears. The samples were stained with hematoxylin and eosin, safranin-O, and Alcian blue (pH 1.0) for light microscopy. Immunohistochemistry was performed using anti-S100, anti-collagen I and II, and anti-tenascin-C antibodies. RESULTS: Histochemistry: LHBB samples showed matrix disorganization, with clusters of chondrocyte surrounded by collagen fibers and glycosaminoglycans. Safranin-O showed evident metachromasia. SHBB samples did not show any matrix disorganization or cartilaginous metaplasia. Immunohistochemistry: In all LHBB samples, anti-S100 and anti-collagen II showed cartilage in proximity of the tendon tear. Tenascin C immunostained closely to the disorganized matrix areas. SHBB, however, showed no positive areas for S-100, anti-collagen II, or tenascin C. CONCLUSIONS: According to our results, we hypothesize that the repeated stimulation in compression may induce the formation of fibrous cartilage. However, to date its role in tendon pathology remains to be clearly defined.

Bone-derived titanium coating improves in vivo implant osseointegration in an experimental animal model.

Coating of orthopaedic or dental Titanium (Ti) implants with extracellular bone matrix components (e.g., Type I collagen or hydroxyapatite) is usually performed to enhance their osseointegration. Aim of the present research is the evaluation of an innovative bone-derived Ti coating, containing bone apatite and Type I bone collagen preserved, in an experimental model. Coated and uncoated titanium implants were inserted into the extra-articular bone of the distal femur of twelve New Zealand White Rabbits. Labelling of bone formation was performed by sequential intraperitoneal administration of three stains. After 45 and 90 days animals were sacrificed. Bone specimens were embedded in a glycol methacrylate resin and sectioned along a plane parallel to the long axis of the implants for histomorphometric, scanning electron microscopy and energy dispersive X-ray analyses. Bone implant contact (BIC), trabecular thickness (Tb.Th) and calcium-phosphorus ratio were measured. Data were subjected to nonparametric Wilcoxon rank-sum test and Student's t test. All implants healed without adverse reactions. After 45 days from implant, significant (p < 0.05) differences in BIC (55.6 ± 17.1% vs. 29.2 ± 20.1%) and Tb.Th (108.7 ± 67.1 µm vs. 66.6 ± 48.6 µm) were observed between coated and uncoated implants. Significant (p < 0.05) differences in BIC (61.3 ± 2.1% vs. 35.7 ± 16.4%) and Tb.Th (211.4 ± 80.8 µm vs. 150.9 ± 61.5 µm) between coated and uncoated implants were also detected after 90 days. No differences were measured in calcium-phosphorous ratio. Our data indicate that Ti integration can be enhanced by the proposed surface coating. This could accelerate stable implant fixation and early or immediate loading of the device.

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration.

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan-chondroitin sulfate loaded with platelet lysate was included in a poly(D,L-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-ß were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan-PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues.

Total knee prosthesis polyethylene wear reduction by a new metal part finishing method.

PURPOSE: The purpose of this study was to assess a new metal component finishing designed to improve total knee prosthesis durability. Wear of ultrahigh molecular-weight polyethylene (UHMWPE), with generation of submicrometer- and micrometer-sized particles, has been associated with osteolysis and artificial joint failure. Wear extent is influenced by several factors, some of which are related to manufacturing. METHODS: UHMWPE wear was assessed in metal prosthesis components finished with the Microloy® technology and in traditionally finished components by wear simulation experiments (pin on disk and knee simulator tests) and analysis of wear debris. RESULTS: Microloy®-finished prosthesis showed a 48.5% reduction in UHMWPE total weight loss compared with traditional components (P=0.002). A statistically significant (P<0.05) reduction of UHMWPE debris were detected from the Microloy®-finished compared with the traditionally finished components. CONCLUSIONS: These findings suggest the Microloy® metal finishing may enhance the long-term performance of knee prostheses.

Purified collagen I oriented membrane for tendon repair: an ex vivo morphological study.

Injured tendons have limited repair ability after full-thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. The multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham-operated controls). Animals were euthanized 1 or 6 months after surgery, and tendons were subjected to histological and Synchrotron Radiation-based Computed Microtomography (SRµCT) examination and 3D structure analysis. Histological and SRµCT findings showed satisfactory graft integration with native tendon. Histological examination also showed ongoing angiogenesis. Adverse side-effects (inflammation, rejection, calcification) were not observed. The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation.

The effects of growth factors on skeletal muscle lesions.

Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for up to 55% of all sports injuries. The phases of the healing process after direct or indirect muscle injury are complex but clearly defined processes comprising well-coordinated steps: degeneration, inflammation, regeneration, and fibrosis. Despite this frequent occurrence and the presence of a body of data on the pathophysiology of muscle injuries, none of the treatment strategies adopted to date have been shown to be really effective in strictly controlled trials. Most current muscle injury treatments are based on limited experimental and clinical data and/or were only empirically tested. Platelet-rich plasma (PRP) is a promising alternative approach based on the ability of autologous growth factors (GFs) to accelerate tissue healing, improve muscular regeneration, increase neovascularization and reduce fibrosis, allowing rapid recovery after muscle lesions. Thus, further experimental studies that include the quantification of specific GFs released by PRP, as well as additional data on angiogenesis, myogenesis and functional recovery are needed to ultimately validate the hypothesis of PRP efficacy in the treatment of muscle lesions and open the way for its wide clinical application.

Matrix-induced autologous chondrocyte implantation (MACI) in the knee.

PURPOSE: Matrix-induced autologous chondrocyte implantation (MACI) has been in use for chondral defect repair since 2000, but to date, only little is known about its histological outcomes in the repair of knee cartilage defects. This prospective multicentre study aims to evaluate (1) the quality of the repair tissue obtained from biopsies taken during second-look arthroscopy and (2) the relationship between the histological outcome, the macroscopic appearance of the repair and the patients' functional status. METHODS: Thirty-three second-look core biopsies from 30 patients treated with MACI were analysed. At the time of biopsy, the surgeon reported the reason for the second-look arthroscopy, the quality of the repair tissue and the patient's functional status on a standardised form. Biopsies together with patient data were sent to our centre to undergo blind histological evaluation and data analysis. RESULTS: The median overall ICRS II histological score of the examined population was 57 (1st-3rd quartile 41-75). According to the ICRS cartilage repair assessment (CRA) arthroscopic evaluation, 10 biopsies (30%) were classified as normal, 17 (51%) as nearly normal, 4 (12%) as abnormal and 2 (6%) as severely abnormal. The histological outcome was not significantly related either to the macroscopic appearance of the lesion or to the patient's functional status at the time of biopsy. CONCLUSIONS: In the examined population, the macroscopic appearance of the repair tissue gave an overly favourable impression in comparison with the real histological composition of the tissue, which was possibly still maturing in many cases. The healing process after MACI needs to be better understood through a larger histological study, and a longer follow-up is needed to better clarify the relationship between histology and long-term functional status. LEVEL OF EVIDENCE: IV.

Development and characterization of rhVEGF-loaded poly(HEMA-MOEP) coatings electrosynthesized on titanium to enhance bone mineralization and angiogenesis.

Osteointegration of titanium implants could be significantly improved by coatings capable of promoting both mineralization and angiogenesis. In the present study, a copolymeric hydrogel coating, poly-2-hydroxyethyl methacrylate-2-methacryloyloxyethyl phosphate (P(HEMA-MOEP)), devised to enhance calcification in body fluids and to entrap and release growth factors, was electrosynthesized for the first time on titanium substrates and compared to poly-2-hydroxyethyl methacrylate (PHEMA), used as a blank reference. Polymers exhibiting negatively charged groups, such as P(HEMA-MOEP), help to enhance implant calcification. The electrosynthesized coatings were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. MG-63 human osteoblast-like cell behaviour on the coated specimens was investigated by scanning electron microscopy, MTT viability test and osteocalcin mRNA detection. The ability of negatively charged phosphate groups to promote hydroxyapatite-like calcium phosphate deposition on the implants was explored by immersing them in simulated body fluid. Similar biological responses were observed in both coated specimens, while calcium-phosphorus globules were detected only on P(HEMA-MOEP) surfaces pretreated with alkaline solution. Testing of the ability of P(HEMA-MOEP) hydrogels to entrap and release human recombinant vascular endothelial growth factor, to tackle the problem of insufficient oxygen and nutrient delivery, suggested that P(HEMA-MOEP)-coated titanium prostheses could represent a multifunctional material suitable for bone restoration applications.

Collagen I membranes for tendon repair: effect of collagen fiber orientation on cell behavior.

Tendons have poor spontaneous regenerative capabilities, and complete regeneration is never achieved despite intensive remodeling. In this in vitro study, we characterized two multilamellar collagen I membranes differing in the arrangement of collagen fiber deposition (oriented vs. nonoriented) and compared their mechanical properties. Human dermal fibroblasts and tenocytes were seeded on the two membranes to evaluate the effect of fiber orientation on cell viability and cytoskeletal organization. Results demonstrate that the multilamellar collagen I membrane with oriented fibers has the better mechanical properties and affords optimum cell proliferation and adhesion. Its fiber arrangement provides an instructive pattern for cell growth and may serve to guide the alignment of cells migrating from the ends of a crushed or frayed tendon to obtain a strong, correctly structured tendon, thus providing a viable clinical option for tendon repair.

Responsiveness of Dupuytren's disease fibroblasts to 5 alpha-dihydrotestosterone.

PURPOSE: We recently showed that androgen receptors are expressed in Dupuytren's contracture. The aim of the present work was to test the responsiveness of Dupuytren's fibroblasts to 5 alpha-dihydrotestosterone (5 alpha-DHT), the active form of testosterone. RESULTS: Cultured palmar fascia cells from 10 patients with Dupuytren's contracture and 4 normal subjects were exposed to 5 alpha-DHT (10 or 100 ng/mL) for 1, 3, 7, and 15 days. Their phenotype was analyzed immunohistochemically for alpha-smooth muscle actin and androgen receptor expression and proliferation rates were studied. RESULTS: At 15 days the higher concentration of 5 alpha-DHT induced an increase in Dupuytren's fibroblast proliferation, whereas anti-alpha-smooth muscle actin exhibited the strongest expression. At the same time point androgen receptor expression decreased with the lower concentration and disappeared altogether with the higher dose of 5 alpha-DHT. CONCLUSIONS: The palmar fascia is a target tissue for androgen action via androgen receptors. Further studies are required to determine whether control of androgen receptor may control the evolution of Dupuytren's disease.

Engineered articular cartilage: influence of the scaffold on cell phenotype and proliferation.

Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histochemical, immunohistochemical and histomorphometric analysis to study phenotype expression and cell adhesion. At 7, 14 and 21 days cell proliferation was studied by incorporation of [3H]-thymidine. Our data evidence that the collagen type influences cell morphology, adhesion and growth; indeed, cellularity and rate of proliferation were significantly higher and cells were rounder on the collagen II matrix than on either of the collagen I matrices. Among the collagen I matrices, we observed a great variability in terms of cell adhesion and proliferation. The present study allowed us to identify one type I collagen matrix and one type II collagen matrix that could be usefully employed as a scaffold for chondrocyte transplantation.