Micro-fragmentation is a valid alternative to cell expansion and enzymatic digestion of adipose tissue for the treatment of knee osteoarthritis: a comparative preclinical study.

PURPOSE: The aim of this study was to compare three procedures to exploit adipose-derived cells for the treatment of osteoarthritis (OA) in a preclinical model, to understand their therapeutic potential and identify the most suitable approach for the clinical application. METHODS: Biological samples from adipose tissue, processed by mechanical micro-fragmentation (MF), enzymatic digestion (SVF) or cell expansion (ADSCs), were first characterized in vitro and then used in vivo in a surgically induced OA rabbit model: Group 1-control group (untreated 12 knees/saline 12 knees), Group 2-MF (24 knees), Group 3-SVF (24 knees), Group 4-ADSCs (24 knees). Macroscopic, histological, histomorphometric, immunohistochemical and blood and synovial fluid analyses were evaluated at 2 and 4 months from the treatments. RESULTS: Samples obtained by the three procedures yielded 85-95% of viable cells. In vivo assessments showed no significant side effects or inflammatory responses after the injection. The macroscopic Hanashi score did not show significant differences among treated groups and controls. The histopathological evaluation of synovial tissues showed lower signs of synovitis for MF, although the semiquantitative analysis (Krenn score) did not reach statistical significance. Instead, MF showed the best results both in terms of qualitative and semi-quantitative evaluations of articular cartilage, with a more uniform staining, a smoother surface and a significantly better Laverty score (p = 0.004). CONCLUSION: MF, SVF, and expanded ADSCs did not elicit significant local or systemic adverse reactions in this preclinical OA model. Among the different methods used to exploit the adipose tissue potential, MF showed the most promising findings in particular in terms of protection of the articular surface from the joint degenerative OA processes. LEVEL OF EVIDENCE: Preclinical animal study.

Microfragmented abdominal adipose tissue-derived stem cells from knee osteoarthritis patients aged 29-65 years demonstrate in vitro stemness and low levels of cellular senescence.

Purpose: To investigate the level of cellular senescence in stem cells derived from microfragmented abdominal adipose tissue harvested from patients with knee osteoarthritis (OA). Methods: Stem cells harvested from microfragmented abdominal adipose tissue from 20 patients with knee OA, aged 29-65 years (mean = 49.8, SD = 9.58), were analysed as a function of patient age and compared with control cells exhibiting signs of cellular senescence. Steady-state mRNA levels of a panel of genes associated with senescence were measured by qPCR. Intracellular senescence-associated proteins p16 and p21, and senescence-associated ß-galactosidase activity were measured by flow cytometry. Cellular proliferation was assessed using a 5-ethynyl-2'-deoxyuridine proliferation assay. Stemness was assessed by stem cell surface markers using flow cytometry and the capacity to undergo adipogenic and osteogenic differentiation in vitro. Results: No correlation was found between cellular senescence levels of the microfragmented adipose tissue-derived stem cells and patient age for any of the standard assays used to quantify senescence. The level of cellular senescence was generally low across all senescence-associated assays compared to the positive senescence control. Stemness was verified for all samples. An increased capacity to undergo adipogenic differentiation was shown with increasing patient age (p = 0.02). No effect of patient age was found for osteogenic differentiation. Conclusions: Autologous microfragmented adipose tissue-derived stem cells may be used in clinical trials of knee OA of patients aged 29-65 years, at least until passage 4, as they show stemness potential and negligible senescence in vitro. Level of Evidence: Not applicable.

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

PURPOSE: To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis. METHODS: Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence-associated ß-galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin-Red-S and Oil-Red-O staining, respectively. RESULTS: Microfragmented AT from 7 patients was cryopreserved for a period of 46-150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p > 0.05). Low levels of senescence-associated ß-galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31-CD34+CD45-CD90+CD146-), pericytes (CD31-CD34-CD45-CD90+CD146+), transitional pericytes (CD31-CD34+CD45-CD90+CD146+), and CD271+ stem cells (CD31-CD45-CD90+CD271+) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (3% (2%)) at passage 4 (p = 0.04). CONCLUSIONS: Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED.

Characterization of Microfragmented Adipose Tissue Architecture, Mesenchymal Stromal Cell Content and Release of Paracrine Mediators.

The use of microfragmented adipose tissue (µFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis (OA), is gaining popularity, following positive results reported in recent case series and clinical trials. Although these outcomes were postulated to rely on paracrine signals, to date, a thorough fingerprint of released molecules is largely missing. The purpose of this study was to first characterize both structure and cell content of unprocessed lipoaspirate (LA) and µFAT, and further identify and frame the array of signaling factors in the context of OA disease, by means of high throughput qRT-PCR for extracellular-vesicle (EV) embedded miRNAs and proteomics for tissue and secreted factors. Cell count showed reduction of blood cells in µFAT, confirmed by histological and flow cytometry analyses, that also showed a conserved presence of structural, endothelial and stromal components and pericytes. In the secretome, 376 and 381 EV-miRNAs in LA and µFAT, respectively, were identified. In particular, most abundant and µFAT upregulated EV-miRNAs were mainly recapitulating those already reported as ASC-EVs-specific, with crucial roles in cartilage protection and M2 macrophage polarization, while only a scarce presence of those related to blood cells emerged. Furthermore, secretome proteomic analysis revealed reduction in µFAT of acute phase factors driving OA progression. Taken together, these results suggest that processing of LA into µFAT allows for removal of blood elements and maintenance of tissue structure and stromal cell populations, and possibly the increase of OA-protective molecular features. Thus, microfragmentation represents a safe and efficient method for the application of adipose tissue properties in the frame of musculoskeletal disorders.

Mechanisms and potential immune tradeoffs of accelerated coral growth induced by microfragmentation.

Microfragmentation is the act of cutting corals into small pieces (~1 cm2) to accelerate the growth rates of corals relative to growth rates observed when maintaining larger-sized fragments. This rapid tissue and skeletal expansion technique offers great potential for supporting reef restoration, yet the biological processes and tradeoffs involved in microfragmentation-mediated accelerated growth are not well understood. Here we compared growth rates across a range of successively smaller fragment sizes in multiple genets of reef-building corals, Orbicella faveolata and Montastraea cavernosa. Our results confirm prior findings that smaller initial sizes confer accelerated growth after four months of recovery in a raceway. O. faveolata transcript levels associated with growth rate include genes encoding carbonic anhydrase and glutamic acid-rich proteins, which have been previously implicated in coral biomineralization, as well as a number of unannotated transcripts that warrant further characterization. Innate immunity enzyme activity assays and gene expression results suggest a potential tradeoff between growth rate after microfragmentation and immune investment. Microfragmentation-based restoration practices have had great success on Caribbean reefs, despite widespread mortality among wild corals due to infectious diseases. Future studies should continue to examine potential immune tradeoffs throughout the microfragmentation recovery period that may affect growout survival and disease transmission after outplanting.

Microfragmented adipose tissue is associated with improved ex vivo performance linked to HOXB7 and b-FGF expression.

INTRODUCTION: Adipose tissue (AT) has become a source of mesenchymal stromal/stem cells (MSC) for regenerative medicine applications, in particular skeletal disorders. Several enzymatic or mechanical procedures have been proposed to process AT with the aim to isolate cells that can be locally implanted. How AT is processed may impact its properties. Thus, we compared AT processed by centrifugation (C-AT) to microfragmentation (MF-AT). Focusing on MF-AT, we subsequently assessed the impact of synovial fluid (SF) alone on both MF-AT and isolated AT-MSC to better understand their cartilage repair mechanisms. MATERIALS AND METHODS: MF-AT and C-AT from the same donors were compared by histology and qRT-PCR immediately after isolation or as ex vivo cultures using a micro-tissue pellet system. The in vitro impact of SF on MF-AT and AT-MSC was assessed by histological staining and molecular analysis. RESULTS: The main AT histological features (i.e., increased extracellular matrix and cellularity) of the freshly isolated or ex vivo-cultured MF-AT persisted compared to C-AT, which rapidly deteriorated during culture. Based on our previous studies of HOX genes in MSC, we investigated the involvement of Homeobox Protein HOX-B7 (HOXB7) and its target basic Fibroblast Growth Factor (bFGF) in the molecular mechanism underlying the improved performance of MF-AT. Indeed, both these biomarkers were more prominent in freshly isolated MF-AT compared to C-AT. SF alone preserved the AT histological features of MF-AT, together with HOXB7 and bFGF expression. Increased cell performance was also observed in isolated AT-MSC after SF treatment concomitant with enhanced HOXB7 expression, although there was no apparent association with bFGF. CONCLUSIONS: Our findings show that MF has a positive effect on the maintenance of AT histology and may trigger the expression of trophic factors that improve tissue repair by processed AT.

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences.

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

Fish predation hinders the success of coral restoration efforts using fragmented massive corals.

As coral reefs continue to decline globally, coral restoration practitioners have explored various approaches to return coral cover and diversity to decimated reefs. While branching coral species have long been the focus of restoration efforts, the recent development of the microfragmentation coral propagation technique has made it possible to incorporate massive coral species into restoration efforts. Microfragmentation (i.e., the process of cutting large donor colonies into small fragments that grow fast) has yielded promising early results. Still, best practices for outplanting fragmented corals of massive morphologies are continuing to be developed and modified to maximize survivorship. Here, we compared outplant success among four species of massive corals (Orbicella faveolata, Montastraea cavernosa, Pseudodiploria clivosa, and P. strigosa) in Southeast Florida, US. Within the first week following coral deployment, predation impacts by fish on the small (<5 cm2) outplanted colonies resulted in both the complete removal of colonies and significant tissue damage, as evidenced by bite marks. In our study, 8-27% of fragments from four species were removed by fish within one week, with removal rates slowing down over time. Of the corals that remained after one week, over 9% showed signs of fish predation. Our findings showed that predation by corallivorous fish taxa like butterflyfishes (Chaetodontidae), parrotfishes (Scaridae), and damselfishes (Pomacentridae) is a major threat to coral outplants, and that susceptibility varied significantly among coral species and outplanting method. Moreover, we identify factors that reduce predation impacts such as: (1) using cement instead of glue to attach corals, (2) elevating fragments off the substrate, and (3) limiting the amount of skeleton exposed at the time of outplanting. These strategies are essential to maximizing the efficiency of outplanting techniques and enhancing the impact of reef restoration.

Advances in nanocrystallography as a proteomic tool.

In order to overcome the difficulties and hurdles too much often encountered in crystallizing a protein with the conventional techniques, our group has introduced the innovative Langmuir-Blodgett (LB)-based crystallization, as a major advance in the field of both structural and functional proteomics, thus pioneering the emerging field of the so-called nanocrystallography or nanobiocrystallography. This approach uniquely combines protein crystallography and nanotechnologies within an integrated, coherent framework that allows one to obtain highly stable protein crystals and to fully characterize them at a nano- and subnanoscale. A variety of experimental techniques and theoretical/semi-theoretical approaches, ranging from atomic force microscopy, circular dichroism, Raman spectroscopy and other spectroscopic methods, microbeam grazing-incidence small-angle X-ray scattering to in silico simulations, bioinformatics, and molecular dynamics, has been exploited in order to study the LB-films and to investigate the kinetics and the main features of LB-grown crystals. When compared to classical hanging-drop crystallization, LB technique appears strikingly superior and yields results comparable with crystallization in microgravity environments. Therefore, the achievement of LB-based crystallography can have a tremendous impact in the field of industrial and clinical/therapeutic applications, opening new perspectives for personalized medicine. These implications are envisaged and discussed in the present contribution.