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.

How do students approach the study of the History of Medicine? Some considerations after the final exams at the first year and fourth year.

BACKGROUND AND AIM: Reports about the teaching of the History of Medicine in universities worldwide can be found easily in medical literature. They are often comparative studies in which the opinions provided by the professors are collected and the teaching programs are compared. Our study focuses instead on the relationship between the students and the discipline, what they look for from it, and how their interest changes with the progress of the course of study. METHODS: The final tests of the students of two Italian universities, Parma and Bologna, were analyzed, in which the candidate had the ability to choose the topic of discussion and to outline his personal analysis. The course year in which the final examination was faced is different: in the first year in Bologna, in the fourth year in Parma. RESULTS: This survey show that in both universities most students have carried out autonomous research regardless of the educational material made available to them. This attitude can be interpreted as a real interest in the history of medicine, widening their search throughout all the fields of the discipline. CONCLUSIONS: These results seem to suggest to teachers of History of Medicine to convey to their students the methodology of historical and epistemological research, giving the student to the pupils the opportunity to become passionate about history in the way he/she prefers. (www.actabiomedica.it).

Meniscectomy-induced osteoarthritis in the sheep model for the investigation of therapeutic strategies: a systematic review.

PURPOSE: One of the major risk factors for OA is meniscectomy (Mx) that causes a rapid and progressive OA. Mx has been employed in various animal models, especially in large ones, to study preclinical safety and strategy effectiveness to counteract OA. The aim of the present study is to review in vivo studies, performed in sheep and published in the last ten years. METHODS: The search strategy was performed in three websites: www.scopus.com, www.pubmed.com, and www.webofknowledge.com, using "Meniscectomy and osteoarthritis in sheep" keywords. RESULTS: The 25 included studies performed unilateral total medial Mx (MMx), unilateral partial MMx, bilateral MMx, unilateral total lateral Mx (LMx), unilateral partial LMx, and bilateral LMx and MMx combined with anterior cruciate ligament transaction. The most frequently performed is the unilateral total MMx that increases changes in cartilage and subchondral bone more than the other techniques. Gross evaluations, histology, radiography, and biochemical tests are used to assess the degree of OA. The most widely tested treatments are related to scaffolds with or without mesenchymal stem cells. CONCLUSION: OA therapeutic strategies require the use of large animal models due to similarities with human joint anatomy. A protocol for future in vivo studies on post-traumatic OA is clarified.

Regenerative Features of Adipose Tissue for Osteoarthritis Treatment in a Rabbit Model: Enzymatic Digestion Versus Mechanical Disruption.

Evaluating cell migration after cell-based treatment is important for several disorders, including osteoarthritis (OA), as it might influence the clinical outcome. This research explores migrating expanded-adipose stromal cells (ASCs) and adipose niches after enzymatic and mechanical processes. Bilateral anterior cruciate ligament transection induced a mild grade of OA at eight weeks in adult male New Zealand rabbits. ASCs, enzymatic stromal vascular fraction (SVF), and micro fragmented adipose tissue (MFAT) were intra-articularly injected in the knee joint. Assessments of cell viability and expression of specific markers, including CD-163 wound-healing macrophages, were done. Cell migration was explored through labelling with PKH26 dye at 7 and 30 days alongside co-localization analyses for CD-146. All cells showed good viability and high percentages of CD-90 and CD-146. CD-163 was significantly higher in MFAT compared to SVF. Distinct migratory potential and time-dependent effects were observed among cell-based treatments. At day 7, both ASCs and SVF migrated towards synovium, whereas for MFAT versus cartilage, a different migration pattern was noticed at day 30. The long-term distinct cell migration of ASCs, SVF, and MFAT open interesting clinical insights on their potential use for OA treatment. Moreover, the highest expression of CD-163 in MFAT, rather than SVF, might have an important role in directly mediating cartilage tissue repair responses.

Auto-Allo Graft Parallel Juxtaposition for Improved Neuroregeneration in Peripheral Nerve Reconstruction Based on Acellular Nerve Allografts.

INTRODUCTION: Nerve repair poses a significant surgical challenge, and much research on this topic for improvement in reconstruction of segmental defects is ongoing. The aims of the study were to reconfirm the accuracy and safety of a previously described nerve decellularization method on a larger experimental population of rabbits, as well as on human nerves, and to establish in vivo the efficacy of a new-concept mixed graft, comprising autologous and acellular nerve allograft components within the same graft. METHODS: Acellular nerve allografts were implanted into tibial nerve defects of 5 rabbits (group A), autografts were implanted, representing the criterion standard, in other 5 animals (group B), and the innovative technique was used in the remaining 5 (group C). Twelve weeks postoperatively, nerve conduction evaluations were performed; animals were euthanatized, and grafts were harvested and morphologically, histomorphometrically, and immunohistochemically analyzed. Eventually, a preliminary in vitro validation of the decellularization method was performed on human nerves from a cadaver. RESULTS: No clinical adverse effect was revealed during all the experimental times. No tissue reaction was observed, and in all groups, regenerated fascicles and bundles were shown by histology. However, both histology and histomorphometry demonstrated a better regenerative efficiency in group C. The morphological evaluation of the human nerve treated with the novel method showed complete decellularization. CONCLUSION: The microsurgical combined model demonstrated a better neuroregeneration than did pure autografts and acellular nerve allografts. The decellularization method seemed effective also on human nerves. Deeper investigations are necessary to further validate and transfer this new encouraging protocol to the clinical arena.

Effects of intra-articular hyaluronic acid associated to Chitlac (arty-duo®) in a rat knee osteoarthritis model.

Among conventional osteoarthritis (OA) treatments, intra-articular (i.a) viscosupplementation with hyaluronic acid (HA) is used to restore joint viscoelasticity. However, the rapid clearance and elimination of HA may limit its application. The aim of this study was to verify the improved efficacy of HA within the joint, using a lactose-modified chitosan (chitlac) as a potentially chondroprotective additive. Four weeks after induction of experimental OA by destabilization of the medial meniscus (DMM), 12-week-old Sprague Dawley male rats (n = 30), received once a week, for three weeks, i.a injections of: (i) HA associated to chitlac (ARTY-DUO®), (ii) HA; and (iii) sodium chloride (NaCl). Five animals for each group were euthanized 4 weeks after the first i.a injection, while the remaining five were euthanized 8 weeks after the first i.a injection. The restoration of physiological joint microenvironment was tested by histology, histomorphometry, immunohistochemistry, and microtomography (micro-CT). At 4 and even more at 8 weeks, histological analysis showed a significant decrease in OARSI and Mankin scores, with weaker matrix metalloproteinase (MMP)-3, MMP-13, and Galectin-3 in ARTY-DUO® group versus NaCl and HA groups. A reduction in Galectin-1 and a stronger Collagen II staining was seen in both ARTY-DUO® and HA versus NaCl. A reduction in Kreen-modified score, for synovium inflammation, was observed in the ARTY-DUO® group. Micro-CT measurements did not shown significant differences between the groups. The present results show that i.a ARTY-DUO® injections produce a significant improvement in knee articular cartilage degeneration and synovium inflammation in a rat model of DMM-induced OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Bone marrow aspirate clot: A technical complication or a smart approach for musculoskeletal tissue regeneration?

One of the methods employed to improve healing of damaged tissues is the use of cellular based therapies. A number of regenerative medicine based strategies, from in vitro expanded mesenchymal stem cells (MSCs) to "one-step" procedures using bone marrow (BM) in toto (BM aspirate; BMA) or BM concentrate (BMC), have been developed. Recently, orthopedic researchers focused their attention on the clinical therapeutic potential of BMC and BMA for musculoskeletal regeneration. BMA is reported as an excellent source of cells and growth factors. However, the quality of BM harvest and aspirate is extremely technique-dependent and, due to the presence of megakaryocytes and platelets, BMA is prone to clot. BMA clot formation is usually considered a complication hampering the procedures on both BMC preparation and MSC expansion. Therefore, different protocols have been developed to avoid and/or degrade clots. However, from a biological point of view there is a strong rationale for the use of BMA clot for tissue engineering strategies. This descriptive systematic literature review summarizes preclinical and clinical studies dealing the use of BMA clot for orthopedic procedures and provided some evidence supporting its use as a cell based therapy for cartilage and bone regeneration. Despite these results, there are still few preclinical and clinical studies that carefully evaluate the safety and efficacy of BMA clot in orthopedic procedures. Thus, implementing biological knowledge and both preclinical and clinical studies could help researchers and clinicians to understand if BMA clots can really be considered a possible therapeutic tool.

Mesenchymal stem cells for tendon healing: what is on the horizon?

Tendon injuries are a noteworthy morbidity but at present there are few effective scientifically proven treatments. In recent decades, a number of new strategies including tissue engineering with mesenchymal stem cells (MSCs) have been proposed to enhance tendon healing. Although MSCs are an interesting and promising approach, many questions regarding their use in tendon repair remain unanswered. This descriptive overview of the literature of the last decade explores the in vivo studies on tendon healing, in small and large animal models, which used MSCs harvested from different tissues, and the state of the art in clinical applications. It was observed that there are still doubts about the optimum amount of MSCs to use and their source and the type of scaffolds to deliver the cells. Thus, further studies are needed to determine the best protocol for MSC use in tendon healing. Copyright © 2016 John Wiley & Sons, Ltd.

Autologous Bone Marrow Concentrate in a Sheep Model of Osteoarthritis: New Perspectives for Cartilage and Meniscus Repair.

INTRODUCTION: Cell-based therapies are becoming a valuable tool to treat osteoarthritis (OA). This study investigated and compared the regenerative potential of bone marrow concentrate (BMC) and mesenchymal stem cells (MSC), both engineered with Hyaff(®)-11 (HA) for OA treatment in a sheep model. METHODS: OA was induced via unilateral medial meniscectomy. Bone marrow was aspirated from the iliac crest, followed by concentration processes or cell isolation and expansion to obtain BMC and MSC, respectively. Treatments consisted of autologous BMC and MSC seeded onto HA. The regenerative potential of bone, cartilage, menisci, and synovia was monitored using macroscopy, histology, immunohistochemistry, and micro-computed tomography at 12 weeks post-op. Data were analyzed using the general linear model with adjusted Sidak's multiple comparison and Spearman's tests. RESULTS: BMC-HA treatment showed a greater repair ability in inhibiting OA progression compared to MSC-HA, leading to a reduction of inflammation in cartilage, meniscus, and synovium. Indeed, the decrease of inflammation positively contributed to counteract the progression of fibrotic and hypertrophic processes, known to be involved in tissue failure. Moreover, the treatment with BMC-HA showed the best results in allowing meniscus regeneration. Minor healing effects were noticed at bone level for both cell strategies; however, a downregulation of subchondral bone thickness (Cs.Th) was found in both cell treatments compared to the OA group in the femur. CONCLUSION: The transplantation of BMC-HA provided the best effects in supporting regenerative processes in cartilage, meniscus, and synovium and at less extent in bone. On the whole, both MSC and BMC combined with HA reduced inflammation and contributed to switch off fibrotic and hypertrophic processes. The observed regenerative potential by BMC-HA on meniscus could open new perspectives, suggesting its use not only for OA care but also for the treatment of meniscal lesions, even if further analyses are necessary to confirm its healing potential at long-term follow-up.

The Role of Detraining in Tendon Mechanobiology.

INTRODUCTION: Several conditions such as training, aging, estrogen deficiency and drugs could affect the biological and anatomo-physiological characteristics of the tendon. Additionally, recent preclinical and clinical studies examined the effect of detraining on tendon, showing alterations in its structure and morphology and in tenocyte mechanobiology. However, few data evaluated the importance that cessation of training might have on tendon. Basically, we do not fully understand how tendons react to a phase of training followed by sudden detraining. Therefore, within this review, we summarize the studies where tendon detraining was examined. MATERIALS AND METHODS: A descriptive systematic literature review was carried out by searching three databases (PubMed, Scopus and Web of Knowledge) on tendon detraining. Original articles in English from 2000 to 2015 were included. In addition, the search was extended to the reference lists of the selected articles. A public reference manager (www.mendeley.com) was adopted to remove duplicate articles. RESULTS: An initial literature search yielded 134 references (www.pubmed.org: 53; www.scopus.com: 11; www.webofknowledge.com: 70). Fifteen publications were extracted based on the title for further analysis by two independent reviewers. Abstracts and complete articles were after that reviewed to evaluate if they met inclusion criteria. CONCLUSIONS: The revised literature comprised four clinical studies and an in vitro and three in vivo reports. Overall, the results showed that tendon structure and properties after detraining are compromised, with an alteration in the tissue structural organization and mechanical properties. Clinical studies usually showed a lesser extent of tendon alterations, probably because preclinical studies permit an in-depth evaluation of tendon modifications, which is hard to perform in human subjects. In conclusion, after a period of sudden detraining (e.g., after an injury), physical activity should be taken with caution, following a targeted rehabilitation program. However, further research should be performed to fully understand the effect of sudden detraining on tendons.

The role of eccentric exercise in sport injuries rehabilitation.

INTRODUCTION: Sports injuries frequently involve tendons, muscles and ligaments. The variable outcome of surgery and medical treatment support early functional treatments. Eccentric exercise (EE) showed effectiveness in the management of Achilles tendinopathy (AT), patellar tendinopathy (PT) and lateral epicondyle tendinopathy (LET). Preliminary results of EE in other tendinopathies and sports injuries suggest its wide prescription in the sport rehabilitation field. SOURCES OF DATA: A comprehensive search of PubMed, Web of Science, the Cochrane Collaboration Database, Physiotherapy Evidence Database (PEDro), Evidence Based Medicine (EBM) Search review, National Guidelines, Scopus and Google Scholar was performed using keywords such as 'eccentric exercise', 'sports injuries rehabilitation', 'tendinopathy', 'hamstrings strain' 'adductor injuries' and 'ACL reconstruction rehabilitation'. AREAS OF AGREEMENT: EE, alone or associated with other therapies, represents a feasible, cost-effective and successful tool in the treatment of well-known targets and might be promising in shoulder tendinopathy, adductor-related groin pain, hamstring strains, and ACL rehabilitation. AREA OF CONTROVERSY: The lack of standardization of protocols, the variable amount, quality and follow-up of studies, the different anatomy and pathophysiology of the therapeutic targets limit the evidence of applicability of EE to sports injuries. GROWING POINTS: The role of pathology and biomechanics in the response to EE should be further investigated. AREAS TIMELY FOR DEVELOPING RESEARCH: New randomized controlled trials should test the effectiveness of standardized EE regimens to various sites of sports injuries.

Clinical use of bone marrow, bone marrow concentrate, and expanded bone marrow mesenchymal stem cells in cartilage disease.

Mesenchymal stem cells (MSCs) from bone marrow (BM) are widely used for bone and less for cartilage tissue regeneration due to their self-renewal and differentiating properties into osteogenic or chondrogenic lineages. This review considers the last decade of clinical trials involving a two-step procedure, by expanding in vitro MSCs from BM, or the so called "one-step" procedure, using BM in toto or BM concentrate, for the regeneration of cartilage and osteochondral tissue defects. The following conclusions were drawn: (1) Cartilage defects that can be repaired by the two-step technique are about twice the size as those where the one-step method is used; (2) the two-step procedure is especially used for the treatment of osteoarthritic lesions, whereas the one-step procedure is used for osteochondral defects; (3) the number of transplanted cells ranges between 3.8×10(6) and 11.2×10(6) cells/mL, and the period of cell culture expansion of implanted MSCs varies widely with regard to the two-step procedure; (4) hyaluronic or collagenic scaffolds are used in all the clinical studies analyzed for both techniques; (5) the follow-up of the two-step procedure is longer than that of the one-step method, despite having a lower number of patients; and, finally, (6) the mean age of the patients (about 39 years old) is similar in both procedures. Clinical results underline the safety and good and encouraging outcomes for the use of MSCs in clinics. Although more standardized procedures are required, the length of follow-up and the number of patients observed should be augmented, and the design of trials should be implemented to achieve evidence-based results.

Microbiological and pharmacological tests on new antibiotic-loaded PMMA-based composites for the treatment of osteomyelitis.

Local antibiotic diffusion in rabbit femurs from two new PMMA-based and nail-shaped composites, enriched with ß-tricalcium phosphate (P-TCP) and BaSO(4) or only with BaSO(4) (P-BaSO(4) ), and soaked in a solution of gentamicin (G) and vancomycin (V) was studied. Nails were implanted into the intramedullary cavity of healthy and osteomyelitic femurs to study the resolution of infection and to quantify the antibiotic penetration into bone by microbiological, pharmacological, and histological tests. A significant progression of osteomyelitis was recorded 7 weeks after MRSA inoculation, whereas no bacteria were found in animals treated with antibiotic-loaded nails as confirmed by microbiology and histology (Smeltzer score). The release of both antibiotics from composites was high and prompt both in healthy and infected bone; the amount of V was higher than that of G in all bone samples. Antibiotics of both composites were still present in bone 3 weeks after nail implantation. The P-BaSO4 composite released a lower amount of antibiotics than did P-TCP. The G-V combination in vivo exerted a synergistic bactericidal effect, which was confirmed by microbiological, histological, and clinical results (no infection). These new porous PMMA composites, soaked in G-V solution in the operating room, might be an effective and useful drug delivery system for osteomyelitis treatment.

Bone regeneration potential of a soybean-based filler: experimental study in a rabbit cancellous bone defects.

Autologous and allogenic bone grafts are considered as materials of choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of alternative biomaterials. The present work compares the bone regeneration potential of a soybean based bone filler (SB bone filler) in comparison to a commercial 50:50 poly(D: ,L: lactide-glycolide)-based bone graft (Fisiograft((R)) gel) when implanted into a critical size defect (6-mm diameter, 10-mm length) in rabbit distal femurs. The histomorphometric and microhardness analyses of femoral condyles 4, 8, 16 and 24 weeks after surgery showed that no significant difference was found in the percentage of both bone repair and bone in-growth in the external, medium and inner defect areas. The SB filler-treated defects showed significantly higher outer bone formation and microhardness results at 24 weeks than Fisiograft((R)) gel (P < 0.05). Soybean-based biomaterials clearly promoted bone repair through a mechanism of action that is likely to involve both the scaffolding role of the biomaterial for osteoblasts and the induction of their differentiation.

From Hippocrates to tissue engineering: surgical strategies in wound treatment.

The history of wound treatment has been virtually the history of surgery for many centuries and also is a history of alliance and conflicts between the physician and nature. The Hippocratic statement about natura medicatrix has been well known since antiquity, but often was neglected. Suppuration was considered a necessary event in the healing process and was elicited by the surgeons with traumatic and painful procedures. The concept of simplicity in treating the wounds was suggested by Teodorico Borgognone and Henry de Mondeville in 13th century and was confirmed only three centuries later by the works of Ambroise Paré and Cesare Magati. The history of wound management has been characterized by empiricism since the 18th century, but it took a physiopathological direction during the 19th century when Virchow investigated tissue reaction to injuries, and Lister introduced antiseptic procedures in surgery. By establishing the basis for a biological method to treat wounds, the seeds were sown to enhance the pathways involved in tissue repair, also with the support of new strategies and technology.

Tissue healing in implants immediately placed into postextraction sockets: a pilot study in a mini-pig model.

OBJECTIVE: A pilot in vivo study was conducted to evaluate (1) the rate of osseointegration at apical, middle, and coronal levels of oral implants immediately installed into fresh extraction sockets; (2) the maturation of the newly formed bone surrounding implants during 60 days of healing; and (3) the epithelium seal development. STUDY DESIGN: The premolars of 8 male adult mini-pigs were extracted at each mandibular site under general anesthesia. In the experimental side, Frialit-2 implants were immediately inserted. The gap between bone and implants ranged between 3 and 6 mm circumferencially. Bone specimens were obtained at 7, 15, 30, and 60 days after surgery for histologic and histomorphometric studies. Bone-to-implant contact (BIC), bone volume, trabecular thickness, number, and separation were recorded. Nonparametric exact tests were used to evaluate data. RESULTS: BIC at the coronal level was observed close to 0% at day 7 and increased up to 60% at day 60 after surgery on an average. BIC increased from 11.7% to 47.38% at middle level and from 53.4% to 67.38% at apical level from day 7 to day 60. With respect to bone maturation, in the earlier stages of healing, many thin trabeculae were observed, which, particularly at coronal level, became significantly fewer and thicker in more advanced stages. At day 60, the features of the bone were similar to those of baseline. The epithelium never migrated more than 1.8 mm apically to the top of the alveolar bone level. CONCLUSION: When implants are placed immediately into fresh extraction sockets, in minipig models osseointegration also occurs without initial bone contact.

In vivo experimental study on bone regeneration in critical bone defects using an injectable biodegradable PLA/PGA copolymer.

OBJECTIVES: An assessment was done of the bone-healing rate after implantation of a polylactide/polyglycolide copolymer (PLA-PGA) 50/50 dispersed in aqueous solution of PGA and dextran, used as bone substitutes in an animal model. STUDY DESIGN: Two groups of 5 rabbits each were used. In both the femoral condyles, a critical size defect of 6x10 mm was made. On the right side PLA/PGA was inserted; the left side remained empty. Thirty and 90 days after surgery the animals were killed. RESULTS: Defects left unfilled showed no spontaneous healing after 30 and 90 days. Sites filled with experimental materials showed new bone ranging between 11.46% and 76.82% after 30 days, and 75.98% and 95.34% after 90 days. Histomorphometry showed an increase in bone maturation between day 30 and 90 in experimental sites. At day 90, no statistical difference was seen as compared to normal bone. CONCLUSION: PLA/PGA copolymer dispersed in hydrosoluble matrix seems to be suitable as osteoconductive material in critical size defects.

Osseointegration of endosseous ceramic implants after postoperative low-power laser stimulation: an in vivo comparative study.

Stimulation with low-power laser (LPL) can enhance bone repair as reported in experimental studies on bone defects and fracture healing. Little data exist concerning the use of LPL postoperative stimulation to improve osseointegration of endosseous implants in orthopaedic and dental surgery. An in vivo model was used for the present study to evaluate whether Ga-Al-As (780 nm) LPL stimulation can improve biomaterial osseointegration. After drilling holes, cylindrical implants of hydroxyapatite (HA) were placed into both distal femurs of 12 rabbits. From postoperative day 1 and for 5 consecutive days, the left femurs of all rabbits were submitted to LPL treatment (LPL group) with the following parameters: 300 J/cm2, 1 W, 300 Hz, pulsating emission, 10 min. The right femurs were sham-treated (control group). Three and 6 weeks after implantation, histomorphometric and microhardness measurements were taken. A higher affinity index was observed at the HA-bone interface in the LPL group at 3 (P<0.0005) and 6 weeks (P<0.001); a significant difference in bone microhardness was seen in the LPL group vs. the control group (P<0.01). These results suggest that LPL postoperative treatment enhances the bone-implant interface.