Marco Viganò

Soft-Focused Extracorporeal Shock Waves Increase the Expression of Tendon-Specific Markers and the Release of Anti-inflammatory Cytokines in an Adherent Culture Model of Primary Human Tendon Cells

Focused extracorporeal shock waves have been found to upregulate the expression of collagen and to initiate cell proliferation in healthy tenocytes and to positively affect the metabolism of tendons, promoting the healing process. Recently, soft-focused extracorporeal shock waves have also been found to have a significant effect on tissue regeneration. However, very few in vitro reports have dealt with the application of this type of shock wave to cells, and in particular, no previous studies have investigated the response of tendon cells to this impulse. We devised an original model to investigate the in vitro effects of soft-focused shock waves on a heterogeneous population of human resident tendon cells in adherent monolayer culture. Our results indicate that soft-focused extracorporeal shock wave treatment (0.17 mJ/mm(2)) is able to induce positive modulation of cell viability, proliferation and tendon-specific marker expression, as well as release of anti-inflammatory cytokines. This could prefigure a new rationale for routine employment of soft-focused shock waves to treat the failed healing status that distinguishes tendinopathies.

Blood management and transfusion strategies in 600 patients undergoing total joint arthroplasty: an analysis of pre-operative autologous blood donation

BACKGROUND Blood loss during total joint arthroplasty strongly influences the time to recover after surgery and the quality of the recovery. Blood conservation strategies such as pre-operative autologous blood donation and post-operative cell salvage are intended to avoid allogeneic blood transfusions and their associated risks. Although widely investigated, the real effectiveness of these alternative transfusion practices remains controversial. MATERIALS AND METHODS The surgery reports of 600 patients undergoing total joint arthroplasty (312 hip and 288 knee replacements) were retrospectively reviewed to assess transfusion needs and related blood management at our institute. Evaluation parameters included post-operative blood loss, haemoglobin concentration measured at different time points, ASA score, and blood transfusion strategies. RESULTS Autologous blood donation increased the odds of receiving a red blood cell transfusion. Reinfusion by a cell salvage system of post-operative shed blood was found to limit adverse effects in cases of severe post-operative blood loss. The peri-operative net decrease in haemoglobin concentration was higher in patients who had predeposited autologous blood than in those who had not. DISCUSSION The strengths of this study are the high number of cases and the standardised procedures, all operations having been performed by a single orthopaedic surgeon and a single anaesthesiologist. Our data suggest that a pre-operative autologous donation programme may often be useless, if not harmful. Conversely, the use of a cell salvage system may be effective in reducing the impact of blood transfusion on a patients physiological status. Basal haemoglobin concentration emerged as a useful indicator of transfusion probability in total joint replacement procedures.

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.

Onsets of complications and revisions are not increased after simultaneous bilateral unicompartmental knee arthroplasty in comparison with unilateral procedures

PurposeThe advantages of simultaneous bilateral procedures in joint arthroplasty have been widely described for in total joint replacements of both the hip and the knee. In contrast, unicompartmental knee arthroplasties, despite their effectiveness in pain relieving and functional improvement, are underinvestigated in these terms. The purpose of this study is to assess the possible benefits and risks of bilateral simultaneous knee replacements, in comparison with unilateral procedures.MethodsA total of 567 surgery reports of bilateral simultaneous (220) or unilateral (347) unicompartmental knee arthroplasties were analysed to collect study parameters. Information like the onset of complications and need for revisions were recorded by phone interview (at least two years after surgery). All surgeries were performed by the same orthopaedic surgeon, assisted by the same anesthesiologist.ResultsComplication and revision rates, as well as the length of hospital stay were similar between the two study groups, while blood and haemoglobin losses, and consequently the use of transfusion of allogeneic and autologous blood units, were higher in the simultaneous bilateral group.ConclusionsSimultaneous bilateral unicompartmental knee arthroplasties could significantly reduce, if both joints are affected, the length of hospital stay and, therefore, patient management costs. At the same time, they do not lead to more frequent revisions or complications. The higher transfusion of allogeneic blood units could be reduced to unilateral surgery levels by the application of currently available protocols of autologous blood reinfusion.

Plasma vitamin D and osteo-cartilaginous markers in Italian males affected by intervertebral disc degeneration: Focus on seasonal and pathological trend of type II collagen degradation

OBJECTIVE To evaluate plasma vitamin D and cross-linked C-telopeptides of type I (CTx-I) and type II (CTx-II) collagen concentrations in males with lumbar intervertebral disc degeneration (IVD) compared to healthy controls. Improved knowledge might suggest to optimize the vitamin D status of IVD patients and contribute to clarify mechanisms of cartilage degradation. METHODS 79 Italian males with lumbar IVD assessed by Magnetic Resonance Imaging (MRI) and 79 age, sex and BMI-matched healthy controls were enrolled. Plasma 25hydroxyvitamin D (25(OH)D), CTx-I and CTx-II were measured by immunoassays. Circannual seasonality, correlation between biomarkers concentrations and clinical variables were assessed. RESULTS Overall subjects 25(OH)D and CTx-II showed month rhythmicity with acrophase in August/September and October/November, and nadir in February/March and April/May, respectively. An inverse correlation between 25(OH)D and CTx-I, and a direct correlation between CTx-II and CTx-I were observed. IVD patients, particularly with osteochondrosis, showed higher CTx-II than healthy controls. CONCLUSIONS Month of sampling may affect plasma 25(OH)D and CTx-II concentrations. The correlation between CTx-I and CTx-II suggests an interplay between the osteo-cartilaginous endplate and the fibro-cartilaginous disc. The results of this study highlighted that osteochondrosis associates with increased cartilaginous catabolism. Vitamin D supplementation seems more necessary in winter for lumbar IVD patients.

Different culture conditions affect the growth of human tendon stem/progenitor cells (TSPCs) within a mixed tendon cells (TCs) population

BackgroundTendon resident cells (TCs) are a mixed population made of terminally differentiated tenocytes and tendon stem/progenitor cells (TSPCs). Since the enrichment of progenitors proportion could enhance the effectiveness of treatments based on these cell populations, the interest on the effect of culture conditions on the TSPCs is growing.In this study the clonal selection and the culture in presence or absence of basic fibroblast growth factor (bFGF) were used to assess their influences on the stemness properties and phenotype specific features of tendon cells.MethodsCells cultured with the different methods were analyzed in terms of clonogenic and differentiation abilities, stem and tendon specific genes expression and immunophenotype at passage 2 and passage 4.ResultsThe clonal selection allowed to isolate cells with a higher multi-differentiation potential, but at the same time a lower proliferation rate in comparison to the whole population. Moreover, the clones express a higher amounts of stemness marker OCT4 and tendon specific transcription factor Scleraxis (SCX) mRNA, but a lower level of decorin (DCN). On the other hand, the number of cells obtained by clonal selection was extremely low and most of the clones were unable to reach a high number of passages in cultures.The presence of bFGF influences TCs morphology, enhance their proliferation rate and reduce their clonogenic ability. Interestingly, the expression of CD54, a known mesenchymal stem cell marker, is reduced in presence of bFGF at early passages. Nevertheless, bFGF does not affect the chondrogenic and osteogenic potential of TCs and the expression of tendon specific markers, while it was able to downregulate the OCT4 expression.ConclusionThis study showed that clonal selection enhance progenitors content in TCs populations, but the extremely low number of cells produced with this method could represent an insurmountable obstacle to its application in clinical approaches. We observed that the addition of bFGF to the culture medium promotes the maintenance of a higher number of differentiated cells, reducing the proportion of progenitors within the whole population. Overall our findings demonstrated the importance of the use of specific culture protocols to obtain tendon cells for possible clinical 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.

Meniscal Repair: Enhancement of Healing Process

The meniscus is a crucial player in knee homeostasis, and its preservation is now considered necessary to obtain satisfactory clinical results, above all in the long-term follow-up to avoid the future onset of arthritis. Most of the current meniscal repair techniques are effective in the vascularized zone of the meniscus but fail to promote healing in the avascular zone. For this reason, aiming for improved healing of the meniscus, in addition to fixation techniques and devices, several biologic adjunctive methods have been proposed. They are focused on the delivery of “factors” or “agents” to promote the tissue healing, so that many more patients might benefit from the procedures for the preservation of the menisci. Beyond the most traditional augmentation techniques such as vascular access channel, trephination, abrasion, synovial flaps, and fibrin glue/clot, more recently growth factors, platelet-rich plasma (PRP), and mesenchymal stem cells (MSCs) have also gained attention due to their ability to improve tissue healing, especially in the avascular zone. In this chapter in vitro and clinical findings concerning these augmentation techniques are described and discussed.