Carlotta Perucca Orfei

Multidifferentiation potential of human mesenchymal stem cells from adipose tissue and hamstring tendons for musculoskeletal cell-based therapy

AIM Adipose-derived stem cells (ASCs) have been deeply characterized for their usefulness in musculoskeletal tissue regeneration; recently, other mesenchymal stem cell (MSC) sources have also been proposed. This study compares for the first time human tendon stem/progenitor cells isolated from hamstring tendons with human ASCs. MATERIALS & METHODS Human TSPCs and ASCs were isolated from hamstring tendon portions and adipose tissue of healthy donors undergoing ACL reconstruction or liposuction, respectively (n = 7). Clonogenic ability, immunophenotype and multi-differentiation potential were assessed and compared. RESULTS Both populations showed similar proliferation and clonogenic ability and expressed embryonic stem cell genes and MSC surface markers. Tendon stem/progenitor cells showed lower adipogenic and osteogenic ability, but after the chondrogenic differentiation, they produced more abundant glycosaminoglycans and expressed higher levels of aggrecan with regards to ASCs. The tenogenic induction with BMP-12 upregulated SCX and DCN gene expression in both populations. CONCLUSION Our results demonstrate that waste hamstring tendon fragments could represent a convenient MSC source for musculoskeletal regenerative medicine.

Mesenchymal stem cells as therapeutic target of biophysical stimulation for the treatment of musculoskeletal disorders

BackgroundMusculoskeletal disorders are regarded as a major cause of worldwide morbidity and disability, and they result in huge costs for national health care systems. Traditional therapies frequently turned out to be poorly effective in treating bone, cartilage, and tendon disorders or joint degeneration. As a consequence, the development of novel biological therapies that can treat more effectively these conditions should be the highest priority in regenerative medicine.Main body of the abstractMesenchymal stem cells (MSCs) represent one of the most promising tools in musculoskeletal tissue regenerative medicine, thanks to their proliferation and differentiation potential and their immunomodulatory and trophic ability. Indeed, MSC-based approaches have been proposed for the treatment of almost all orthopedic conditions, starting from different cell sources, alone or in combination with scaffolds and growth factors, and in one-step or two-step procedures. While all these approaches would require cell harvesting and transplantation, the possibility to stimulate the endogenous MSCs to enhance their tissue homeostasis activity represents a less-invasive and cost-effective therapeutic strategy. Nowadays, the role of tissue-specific resident stem cells as possible therapeutic target in degenerative pathologies is underinvestigated. Biophysical stimulations, and in particular extracorporeal shock waves treatment and pulsed electromagnetic fields, are able to induce proliferation and support differentiation of MSCs from different origins and affect their paracrine production of growth factors and cytokines.Short conclusionsThe present review reports the attempts to exploit the resident stem cell potential in musculoskeletal pathologies, highlighting the role of MSCs as therapeutic target of currently applied biophysical treatments.

Fabrication of Innovative Silk/Alginate Microcarriers for Mesenchymal Stem Cell Delivery and Tissue Regeneration

The aim of this study was to exploit silk fibroin’s properties to develop innovative composite microcarriers for mesenchymal stem cell (MSCs) adhesion and proliferation. Alginate microcarriers were prepared, added to silk fibroin solution, and then treated with ethanol to induce silk conformational transition. Microcarriers were characterized for size distribution, coating stability and homogeneity. Finally, in vitro cytocompatibility and suitability as delivery systems for MSCs were investigated. Results indicated that our manufacturing process is consistent and reproducible: silk/alginate microcarriers were stable, with spherical geometry, about 400 μm in average diameter, and fibroin homogeneously coated the surface. MSCs were able to adhere rapidly onto the microcarrier surface and to cover the surface of the microcarrier within three days of culture; moreover, on this innovative 3D culture system, stem cells preserved their metabolic activity and their multi-lineage differentiation potential. In conclusion, silk/alginate microcarriers represent a suitable support for MSCs culture and expansion. Since it is able to preserve MSCs multipotency, the developed 3D system can be intended for cell delivery, for advanced therapy and regenerative medicine applications.

High levels of circulating type II collagen degradation marker (CTx-II) are associated with specific VDR polymorphisms in patients with adult vertebral osteochondrosis

Both vitamin D and collagen have roles in osteocartilaginous homeostasis. We evaluated the association between the circulating 25-hydroxyvitamin D (25(OH)D), type I and II collagen degradation products (CTx-I, and CTx-II), and four vitamin D receptor gene (VDR) polymorphisms, in Italian males affected by low back pain (LBP) due to herniation/discopathy and/or vertebral osteochondrosis. FokI, BsmI, ApaI, and TaqI VDR-polymorphisms were detected through PCR–restriction fragment length polymorphism (RFLP), and circulating 25(OH)D, CTx-I and CTx-II were measured by immunoassays in 79 patients (of which 26 had osteochondrosis) and 79 age-, sex- and body mass index (BMI)-matched healthy controls. Among all 158 subjects, carriers of FF and Ff genotypes showed lower 25(OH)D than ff, which suggested a higher depletion of vitamin D in F allele carriers. Higher CTx-I concentrations were observed in TT versus Tt among controls, and Tt versus tt among LBP cases, which suggested a higher bone-cartilaginous catabolism in subjects bearing the T allele. Higher CTx-II concentrations were observed in patients with osteochondrosis bearing FF, bb, TT, or Aa genotypes in comparison with hernia/discopathy patients and healthy controls. Vertebral osteochondrosis shows peculiar genotypic and biochemical features related to vitamin D and the osteocartilaginous metabolism. Vitamin D has roles in the pathophysiology of osteochondrosis.

Validation of reference and identity-defining genes in human mesenchymal stem cells cultured under unrelated fetal bovine serum batches for basic science and clinical application

The molecular profile of human mesenchymal stem cells (MSCs) have emerged as a key factor in defining their identity. Nevertheless, the effect of fetal bovine serum (FBS) batches or origin on MSC molecular signature has been neglected. In this frame, chemical fingerprint of FBS batches from unrelated countries showed strong correlation between chemical composition and country of origin. Thus, the aim of this study was to evaluate in stem cells isolated from bone marrow (BMMSCs) and umbilical cord-blood (CBMSCs) the effects of independently collected FBS batches on both twelve commonly used reference genes (RGs) and a selected panel of thirty-eight genes crucial for MSC definition in both research and clinical settings. Gene expression stability was estimated comparing the outcomes of two applets: geNorm and NormFinder. The bioinformatics analysis emphasized that, in a panorama of general balance, few RG candidates (YWHAZ/UBC for BMMSCs, RPLP0/EF1A for CBMSCs and EF1A/TBP for both MSCs scored together) showed superior stability. In addition, a wider study on genes involved in differentiation/proliferation/stemness processes, often used to define MSC potency, showed that these genes exhibited no major transcriptional modulation after treatment with different FBS, and allowed the identification of genes strongly discriminating between BM- and CBMSC populations. Therefore, in conclusion, FBS origin does not dramatically impact the general molecular profile of MSCs, although we could identify validated candidates able to allow more reliable comparison of data regarding MSC identity and potency and obtained by research laboratories and clinical manufacturers using different sera.

Silk/Fibroin Microcarriers for Mesenchymal Stem Cell Delivery: Optimization of Cell Seeding by the Design of Experiment

In this methodological paper, lyophilized fibroin-coated alginate microcarriers (LFAMs) proposed as mesenchymal stem cells (MSCs) delivery systems and optimal MSCs seeding conditions for cell adhesion rate and cell arrangement, was defined by a Design of Experiment (DoE) approach. Cells were co-incubated with microcarriers in a bioreactor for different time intervals and conditions: variable stirring speed, dynamic culture intermittent or continuous, and different volumes of cells-LFAMs loaded in the bioreactor. Intermittent dynamic culture resulted as the most determinant parameter; the volume of LFAMs/cells suspension and the speed used for the dynamic culture contributed as well, whereas time was a less influencing parameter. The optimized seeding conditions were: 98 min of incubation time, 12.3 RPM of speed, and 401.5 µL volume of cells-LFAMs suspension cultured with the intermittent dynamic condition. This DoE predicted protocol was then validated on both human Adipose-derived Stem Cells (hASCs) and human Bone Marrow Stem Cells (hBMSCs), revealing a good cell adhesion rate on the surface of the carriers. In conclusion, microcarriers can be used as cell delivery systems at the target site (by injection or arthroscopic technique), to maintain MSCs and their activity at the injured site for regenerative medicine.

The Effect of Three Different Suture Anchors for Rotator Cuff Repair on Primary Cultures of Human Bone Marrow Mesenchymal Stem Cells

Purpose  The purpose of this study is to investigate the in vitro biocompatibility of three different suture anchors (all-suture anchor, metal anchor, and polyetheretherketone anchor), commonly used for the rotator cuff repair. Methods  To assess the biocompatibility of the anchors, the possible cytotoxicity and the immunogenicity of the devices were assessed by cell viability assay and cell count on cultures of bone marrow stem cells (BMSCs) and peripheral blood leucocytes (PBLs), respectively. The possible inhibitory effect of the devices on BMSCs osteogenic potential was evaluated by alkaline phosphatase activity and matrix deposition assay. Results  The viability of BMSCs was slightly reduced when cultured in the presence of the devices (−24 ± 3%). Nevertheless, they were able to differentiate toward the osteogenic lineage in all culture conditions. The proliferation of PBLs and the production of interleukin-2 were not enhanced by the presence of any device. Conclusion  The analyzed devices did not significantly affect the normal cells functions when directly cultured with human primary BMSCs or PBLs, in terms of osteogenic differentiation and inflammatory reaction. Clinical Relevance  A deeper knowledge of the biological reactions to different devices used in rotator cuff surgeries would improve the clinical outcome of these procedures.

Vitamin D’s Effect on the Proliferation and Inflammation of Human Intervertebral Disc Cells in Relation to the Functional Vitamin D Receptor Gene FokI Polymorphism

Vitamin D is known to have immunomodulatory effects, is involved in osteo-cartilaginous metabolism, and may have a role in human intervertebral disc pathophysiology. Although a link between vitamin D receptor (VDR) gene variants and disc degeneration-related pathologies has been observed, its functional contribution to pathologic processes has not been assessed yet. The aim of this study was to investigate the response of disc cells to vitamin D in terms of the regulation of proliferation, metabolism, and inflammatory processes, with a particular focus on the FokI VDR genotype. However, although it was found that vitamin D had a pro-apoptotic effect regardless of genotype, an up-regulation of IL-1Ra and downregulation of IL-6 was found to be evident only in Ff cells. Regarding the metabolic effects, in Ff cells, vitamin D promoted an upregulation of the aggrecan in inflammatory conditions but did not have an effect on the expression of collagen-related markers. Moreover, cells bearing the Ff genotype were the most responsive to vitamin D in the upregulation of catabolic markers. In addition, in contrast to the FF genotype, vitamin D downregulated the vitamin D-dependent signaling pathway in inflamed Ff cells, counteracting the inflammation-mediated catabolic effects. In conclusion, Ff cells were found to be more responsive to the anti-inflammatory and catabolic effects of vitamin D, which is likely to be related to matrix remodeling.

Gene Therapy, Growth Factors, Mesenchymal Cells, New Trends and Future Perspectives

Meniscal tears are common, prevalent intra-articular knee injury and the most frequent cause of orthopedic surgical procedures [1], being a significant risk factor for the development of osteoarthritis (OA) [2]. As widely documented in the previous chapters, tears in the peripheral (vascularized) portion of the meniscus can be repaired using a variety of operative procedures, while those in the central (avascular) area have a poorer healing capacity. Reconstruction of a torn meniscus in this location is challenging, and the long-term effect of allografts on the progression of OA remains uncertain [3].

Treatment of Achilles Tendinopathy with Autologous Adipose-derived Stromal Vascular Fraction Results of a Randomized Prospective Clinical Trial

Objectives: Achilles tendinopathy commonly occurs in both active and inactive persons. It consists in the development of pain and inflammation in the early phases, with progression to the development of fibrotic tissue and degeneration of tendon matrix. Current conservative treatment approaches do not provide sustained satisfactory results, particularly in active patients, although platelet rich plasma (PRP) injection have shown to be effective in many cases. The therapeutic effect of adipose-derived mesenchymal stem cells (ASCs), either expanded or used directly within the stromal vascular fraction (SVF), have demonstrated to possess significant anti-inflammatory and immunomodulatory effects, mediated by the release of active factors, and thus potentially useful in the treatment of tendinopathy. Methods: Patients affected by non-insertional Achilles tendinopathy (range 18-55 y/o) were prospectively enrolled in this controlled study, and randomly assigned either to single PRP injection group (GPSIII kit, Biomet, USA) (n=28 tendons) or single adipose tissue SVF (FastKit, Corios, Italy) (n=28 tendons) injection group. All patients were assessed clinically pre-operatively and at 15, 30, 60, 120 and 180 days from treatment, using VAS Pain, VISA-A, AOFAS and SF-36 forms. Patients also underwent to US and MRI before treatment and then at 4 and 6 month-follow-ups. An aliquot of SVF of each patient was analyzed in vitro for mesenchymal stem cells (MSC) content, viability, proliferation rate, differentiation potential and immunomodulatory ability. Sample size of the study was calculated with a power analysis based on VISA-A score. All the results are expressed as mean ± standard deviation. A Wilcoxon test for paired data was performed to compare variables before and after surgery. Results: Population background data and pre-operative scores were similar in the two groups (p>0.05). At final follow up both patients group showed significantly improvements in all the scores in comparison to baseline (p<0.05). In SVF patients these improvements were faster, with significantly better scores with respect to pre-injection level already starting 15 days after treatment. Indeed at this time point a significant difference between groups in term of VAS, AOFAS and VISA-A score was observed (p<0.05), with better results in the SVF group. After 6 months MR and ultrasounds showed an improvement of clinical signs in both groups, without relevant differences. No side effects were observed in neither groups. In vitro analysis showed a modest content of MSCs in the SVF samples; however these cells were able to efficiently proliferate and differentiate, and to exert good immunomodulatory effect in an in vitro inflammatory model. Conclusion: Both PRP and SVF are safe and effective treatments for Achilles tendinopathy. However, SVF allowed to obtain faster results, thus allowing to consider this treatment particularly suitable for patients requiring to come back to physical activities sooner.