Sandra Manzotti

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-β 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.

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.

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.

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.

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

PurposeMatrix-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.MethodsThirty-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.ResultsThe 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.ConclusionsIn 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.

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.

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.

Responsiveness of Dupuytren’s disease fibroblasts to 5α-dihydrotestosterone

Abstract Purpose We recently showed that androgen receptors are expressed in Dupuytrens contracture. The aim of the present work was to test the responsiveness of Dupuytrens fibroblasts to 5α-dihydrotestosterone (5α-DHT), the active form of testosterone. Methods Cultured palmar fascia cells from 10 patients with Dupuytrens contracture and 4 normal subjects were exposed to 5α-DHT (10 or 100 ng/mL) for 1, 3, 7, and 15 days. Their phenotype was analyzed immunohistochemically for α-smooth muscle actin and androgen receptor expression and proliferation rates were studied. Results At 15 days the higher concentration of 5α-DHT induced an increase in Dupuytrens fibroblast proliferation, whereas anti-α-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 5α-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 Dupuytrens disease.

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 Students 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.

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.