Bilayer scaffolds and tSVF to enhance osteochondral repair

Osteochondral defects still challenge orthopaedic surgeons. It is currently possible to address chondral lesions with a variety of treatment options, though they do not always lead to highly satisfying outcomes. Even more so, there is limited choice in treating osteochondral lesions and either simple, low-cost techniques such as bone marrow stimulation, or more complex and expensive alternatives, have shown to be of limited effectiveness in long-term follow-ups or linked with downsides, such as donor-site morbidity for autologous osteochondral transplantation. Recently, thanks to the progresses in the biomaterial field, an increasing interest have been raised by the healing potential of combining scaffolds and stem cells. Currently, a variety of different natural or synthetic materials have been used to develop scaffolds. Scaffolds have been tested both empty or embedded with growth factors and cells, either autologous chondrocytes, or BMSCs or ADSCs. All of these cell populations need to be harvested from the patient and cultured and expanded in vitro before being seeded on the scaffolds. These techniques therefore necessitate two-step surgeries. The aim of this study was to assess the feasibility of using the whole tissue-Stromal Vascular Fraction as a source of stem cell for bilayer scaffold-based osteochondral lesion repair. The potential main advantage of this technique would be a one-step surgery, in which the adipose tissue is firstly harvested, then processed to obtain tSVF and finally seeded on the osteochondral scaffold. In the first phase of the study, three different processing systems to achieve tSVF from lipoaspirate (micro-fragmentation, filtration, or slow centrifugation) have been evaluated in terms of cell proliferation in vitro and clinical results of intraarticular injections for the treatment of knee OA. From December 2017 to June 2018, 25 procedures were performed using three different systems. A considerable improvement of the clinical condition in almost all patients already one month after the treatment with a stable effect at 6 and 12 months was recorded. Patients treated with SVF, obtained by the micro-fragmentation system, had better outcomes one month after the treatment with a mean improvement of the symptomatology higher than that found in patients treated with the filtration or slow centrifugation system. The SVF product from the same system had a higher cell proliferation capacity in vitro. In the second phase, the micro-fragmentation method was compared to a newer mechanical processing method, the modified-Nanofat grafting. In addition, ADSCs culture-expanded and tSVF, obtained by the two different processing methods, were seeded on bilayer scaffolds to evaluate their cell viability. The main finding of this study was that the modified Nanofat and the micro-fragmentation methods to obtain tSVF both lead to a population of adult Adipose-Derived Stem Cells which showed gene expression, stem cells CD markers and differentiation potential similar to ADSCs cultured expanded obtained by the traditional and well-established enzymatic collagenase-based digestion. Analyzed cells from this study were positive for NANOG and SOX2 expression, confirming that all the processing methods yielded to ADSCs. It was noted a trend towards a higher expression of NANOG by enzymatic-digestion derived ADSCs compared to mechanically methods. Findings of cytofluorometry analysis from this study showed as cultured expanded cells from either enzymatic digestion or mechanical processing expressed the correct CD markers to be defined ADSCs (CD90, CD105 and CD73 positive, CD45 negative). Cell seeding of cultured expanded ADSCs on bilayer demonstrated good cell viability by the live/dead assay. In conclusion, it can be said that micro-fragmentation and modified-Nanofat methods yield to a population of cells which is reach in ADSCs (CD90+, CD73+, CD105+ and CD45-), which express genes characteristic of stemness and which showed differentiation potential towards osteogenic and chondrogenic tissues. A clear superiority of one mechanical method over the other was not found, although cells from modified-Nanofat showed a tendency towards a higher expression of SOX2, however this result has to be further investigated. It was not possible to establish the feasibility of culturing the whole tSVF of a bilayer osteo-chondrogenic scaffolds, therefore further research is needed.