Prospects on the Potential In Vitro Regenerative Features of Mechanically Treated-Adipose Tissue for Osteoarthritis Care.

Gathering a better grasp on the adipose stromal vascular fraction (SVF) is demanding among clinicians for osteoarthritis (OA) care because of its promising but multifaceted clinical outcomes. The aim of this preclinical in vitro study was to test whether the mechanical approach with Hy-Tissue SVF system, a class IIa CE marked device of adipose tissue micro-fragmentation, influences the biological features and functions of SVF. We compared mechanical generated-SVF (mSVF) with the enzymatic generated-SVF (eSVF) by testing cell survival, phenotype, differentiation, and paracrine properties using ELISA assays. Both adipose SVF showed 80% viable cells and enrichment for CD-44 marker. The mSVF product preserved the functions of cell populations within the adipose tissue; however, it displayed lowered nucleated cell recovery and CFU-F than eSVF. As for multipotency, mSVF and eSVF showed similar differentiation commitment for osteochondral lineages. Both adipose SVF exhibited an increased release of VEGF, HGF, IGF-1 and PDGF-bb, involved in pathways mediating osteochondral repair and cell migration. Both mSVF and eSVF also displayed high release for the anti-inflammatory cytokine IL-10. After in vitro culture, supernatants from both mSVF and eSVF groups showed a low release of cytokines except for IL-10, thereby giving evidence of functional changes after culture expansion. In this study, mSVF showed active cell populations in the adipose tissue comparable to eSVF with excellent survival, differentiation and paracrine properties under a new mechanical adipose tissue micro-fragmentation system; thereby suggesting its potential use as a minimally invasive technique for OA treatment.

Subchondral bone response to injected adipose-derived stromal cells for treating osteoarthritis using an experimental rabbit model.

Although articular cartilage is the target of osteoarthritis (OA), its deterioration is not always clearly associated with patient symptoms. Because a functional interaction between cartilage and bone is crucial, the pathophysiology of OA and its treatment strategy must focus also on subchondral bone. We investigated whether adipose-derived stromal cells (ASCs) injected into a joint at two different concentrations could prevent subchondral bone damage after the onset of mild OA in a rabbit model. We measured both volumetric and densitometric aspects of bone remodeling. Although OA can stimulate bone remodeling either catabolically or anabolically over time, the accelerated turnover does not allow complete mineralization of new bone and therefore gradually reduces its density. We measured changes in morphometric and densitometric bone parameters using micro-CT analysis and correlated them with the corresponding parameters in cartilage and meniscus. We found that ASCs promoted cartilage repair and helped counteract the accelerated bone turnover that occurs with OA.

Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

Failures in bipolar fresh osteochondral allograft for the treatment of end-stage knee osteoarthritis.

PURPOSE: Bipolar fresh osteochondral allografts (BFOA) recently became a fascinating option for articular cartilage replacement, in particular in those young patients non-suitable for traditional replacement because of age. While the use of osteochondral allografts for the treatment of focal osteochondral lesions in the knee is well established, their use in the treatment of end-stage arthritis is far more controversial. The purpose of this paper is to describe our experience in a series of seven patients who underwent a resurfacing of both tibio-femoral and patello-femoral joints by BFOA. METHODS: From 2005 to 2007, seven patients (mean age 35.2 ± 6.3 years) underwent BFOA for end-stage arthritis of the knee. Patients were evaluated clinically, radiographically and by CT scan preoperatively and at established intervals up to the final follow-up. RESULTS: No intra-operative complications occurred. Nevertheless, joint laxity and aseptic effusion, along with a progressive chondrolysis, lead to early BFOA failure in six patients, which were revised by total knee arthroplasty at 19.5 ± 3.9 months follow-up. Only one patient, who received the allograft to convert a knee arthrodesis, gained a satisfactory result at the last follow-up control. CONCLUSIONS: BFOA in the knee joint still remains an inapplicable option in the treatment of post-traumatic end-stage arthritis of the young patient, due to the high rate of failure. Further studies are necessary in order to investigate the causes of failure and improve the applicability of this method. Still, after extensive counselling with the patient, BFOA may represent a salvage procedure aimed to revise scarcely tolerated knee arthrodesis. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Bone marrow concentrated cell transplantation: rationale for its use in the treatment of human osteochondral lesions.

Bone marrow is one of the best characterized stem cell microenvironments that contains Mesenchymal Stem Cells (MSCs), a rare population of non-hematopoietic stromal cells. MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages such as bone, cartilage and adipose tissue. However, isolation procedures are crucial for the functional activity of the transplanted cells. The use of concentrated bone marrow cells (BMCs) enables a cell population surrounded by its microenvironment (niche) to be implanted while avoiding all the complications related to the in vitro culture. The cells of the niche are able to regulate stem cell behavior through direct physical contact and secreting paracrine factors. The aim of this study was to characterize BMCs in vitro to evaluate their ability to differentiate toward mature cells and try to understand whether there are differences in the chondrogenic and osteogenic potential of cells from patients of different ages. Mononuclear Cells (MNCs) isolated by Ficoll were used as control. Both cell populations were grown in monolayers and differentiated with specific factors and analyzed by histological and molecular biology assays to evaluate the expression of some specific extracellular matrix molecules. The present investigations revealed the ability of BMCs to act as isolated cells. They are able to form colonies and differentiate toward chondrogenic and osteogenic lineages, the latter pathway appearing to be influenced by donor age. The results obtained by this study support the use of BMCs in clinical practice for the repair of osteochondral damage, which might be particularly useful for the one-step procedure allowing cells to be directly implanted in operating room.

Treatment of human cartilage defects by means of Nd:YAG Laser Therapy.

Articular cartilage lesions represent a challenging problem for orthopaedic surgeons. The purpose of this study was to evaluate the effect of a new pulsed Nd:YAG High Intensity Laser Therapy on the regeneration of cartilage tissue in patients with traumatic lesions. Clinical, histological and immunohistochemical evaluations were performed. Ten patients affected by chondral lesions scheduled for ACI procedure, were enrolled into the study. During the chondrocyte expansion for ACI procedure, cartilage from five patients was treated by Nd:YAG High Intensity Laser Therapy (HILT group). No laser treatment was performed in the remaining patients, who were used as controls. Cartilage repair was assessed by clinicians using two different scores: Cartilage Repair Assessment (CRA) and Overall Repair Assessment (ORA). Cartilage biopsy specimens were harvested to perform histological and immunohistochemical analyses at T0 (before laser treatment) and T1 (at the end of the treatment). A significant decrease in cartilage depth was noticed in the HILT group at T1. Histological and immunohistochemical evaluations showed some regenerative processes in cartilaginous tissue in terms of high amount of proteoglycans, integration with adjacent articular cartilage and good cellular arrangement in the HILT group. By contrast, a not well organized cartilaginous tissue with various fibrous features in the control group at T0 and T1 was observed. In conclusion, the use of this new pulsed Nd:YAG HILT resulted promising in the treatment of moderate cartilage lesions markedly in the young patients.

Evaluation of chondrocyte behavior in a new equine collagen scaffold useful for cartilage repair.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesions: technique note and an early stability pilot clinical trial.

INTRODUCTION: Osteochondral articular defects are a key concern in orthopaedic surgery. Current surgical techniques to repair osteochondral defects lead to poor subchondral bone regeneration and fibrocartilage formation, which is often associated with joint pain and stiffness. The objective of this pilot clinical study is to evaluate the performance and the intrinsic stability of a newly developed biomimetic osteochondral scaffold and to test the safety and the feasibility of the surgical procedure. METHODS: A gradient composite osteochondral scaffold based on type I collagen-hydroxyapatite was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. Thirteen patients (15 defect sites) were treated with scaffold implantation from January 2007 to July 2007: four at the medial femoral condyle, two at the lateral femoral condyles, five at the patellas and four at the trochleas. The mean size of the defects was 2.8 cm(2) (range: 1.5-5.9 cm(2)). All patients were followed up prospectively. High-resolution magnetic resonance imaging (MRI) was used to determine "the early postoperative adherence rate" at 4-5 weeks and 25-26 weeks after scaffold implantation. Moreover, the magnetic resonance observation of cartilage repair tissue (MOCART) score was performed on every MRI. Two second-looks were performed at 6 months; cartilage repair was assessed using the International Cartilage Repair Society (ICRS) visual scoring system and histological and immunohistochemical analysis of the two biopsies was carried out. RESULTS: A completely attached graft and repair tissue were found in 13 of 15 lesions (86.7%). A partial detachment was observed in two patients (13.3%). No detached grafts were found. Complete filling of the cartilage defect and congruency of the articular surface were seen in 10 lesions (66.7%) with MRI evaluation at 6 months. The complete integration of the grafted cartilage was detected in eight lesions (53.3%). Subchondral bone changes (oedema or sclerosis) were found in eight defects (53.3%). Statistical analysis showed a significant improvement in the International Knee Documentation Committee (IKDC) subjective and objective scores from preoperative to 6 months' follow-up (p<0.0005). Visual scoring of the repaired tissue at second-look revealed a normal repair score in one case and a near-normal repair score in the other case. Histological analysis showed the formation of subchondral bone without the presence of biomaterial. The cartilage repair tissue appeared to be engaged in an ongoing maturation process. CONCLUSIONS: The technique is safe and MRI evaluation at short-term follow-up has demonstrated good stability of the scaffold without any other fixation device. The preliminary clinical results at short-term follow-up are encouraging. A clinical and MRI study with longer follow-up and randomised studies will be done to confirm the high potential of this novel osteochondral scaffold.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

A molecular and histological characterization of cartilage from patients with Morquio syndrome.

OBJECTIVE: To investigate the gene expression profile and the histological aspects of articular cartilage of patients affected by Morquio syndrome, a lysosomal storage disease characterized by the accumulation of glycosaminoglycans within the cells which result in abnormal formation and growth of the skeletal system. METHOD: Articular cartilage samples were obtained from the femoral condyle of two siblings with Morquio syndrome during surgery performed to treat valgus knee. As controls, four biopsy samples of healthy cartilage were obtained from four different male multiorgan donors. A Real-Time Polymerase Chain reaction (RT-PCR) analysis was performed to evaluate the expression of type I and II collagens and aggrecan mRNAs. Histological and immunohistochemical analyses for some matrix proteins were carried out on paraffin embedded sections. RESULTS: Type I collagen mRNA mean level was higher in the samples of patients with Morquio syndrome compared to controls. Type II collagen and aggrecan mRNAs' mean expression was instead lower. The morphological appearance of the cartilage showed a poorly organized tissue structure with not homogeneously distributed cells that were larger compared to normal chondrocytes due to the presence inside the vacuoles of proteoglycans which were not metabolized. Chondrocytes were negative for collagen II immunostaining while the extracellular matrix was weakly positive. Collagen type I immunostaining was positive at cellular level. Keratan sulfate showed diffuse positivity and chondroitin-6-sulfate was present throughout the cartilaginous thickness. CONCLUSION: In cartilage of patients with Morquio syndrome, a low expression of collagen type II and a high expression of collagen type I both at protein and molecular levels are evidentiated. This finding could give evidence of the reduction in ankle and knee joint movement observable in these patients.