Exploring In Vivo Models of Musculoskeletal Frailty: A Comprehensive Systematic Review.

Musculoskeletal frailty-a common and debilitating condition linked to aging and chronic diseases-presents a major public health issue. In vivo models have become a key tool for researchers as they investigate the condition's underlying mechanisms and develop effective interventions. This systematic review examines the current body of research on in vivo models of musculoskeletal frailty, without any time constraints. To achieve this aim, we utilized three electronic databases and incorporated a total of 11 studies. Our investigation delves into varied animal models that simulate specific features of musculoskeletal frailty, including muscle loss, bone density reduction, and functional decline. Furthermore, we examine the translational prospects of these models in augmenting our comprehension of musculoskeletal frailty and streamlining the production of groundbreaking therapeutic approaches. This review provides significant insights and guidance for healthcare researchers and practitioners who aim to combat musculoskeletal frailty, ultimately enhancing the quality of life for older adults and individuals affected by this condition.

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

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.

Naturally Occurring Osteoarthritis Features and Treatments: Systematic Review on the Aged Guinea Pig Model.

To date, several in vivo models have been used to reproduce the onset and monitor the progression of osteoarthritis (OA), and guinea pigs represent a standard model for studying naturally occurring, age-related OA. This systematic review aims to characterize the guinea pig for its employment in in vivo, naturally occurring OA studies and for the evaluation of specific disease-modifying agents. The search was performed in PubMed, Scopus, and Web of Knowledge in the last 10 years. Of the 233 records screened, 49 studies were included. Results showed that within a relatively short period of time, this model develops specific OA aspects, including cartilage degeneration, marginal osteophytes formation, and subchondral bone alterations. Disease severity increases with age, beginning at 3 months with mild OA and reaching moderate-severe OA at 18 months. Among the different strains, Dunkin Hartley develops OA at a relatively early age. Thus, disease-modifying agents have mainly been evaluated for this strain. As summarized herein, spontaneous development of OA in guinea pigs represents an excellent model for studying disease pathogenesis and for evaluating therapeutic interventions. In an ongoing effort at standardization, a detailed characterization of specific OA models is necessary, even considering the main purpose of these models, i.e., translatability to human OA.

Benefits of Applying Nanotechnologies to Hydrogels in Efficacy Tests in Osteoarthritis Models-A Systematic Review of Preclinical Studies.

Osteoarthritis (OA) is a severe musculoskeletal disease with an increasing incidence in the worldwide population. Recent research has focused on the development of innovative strategies to prevent articular cartilage damage and slow down OA progression, and nanotechnologies applied to hydrogels have gained particular interest. The aim of this systematic review is to investigate the state of the art on preclinical in vitro and in vivo efficacy studies applying nanotechnologies to hydrogels in OA models to elucidate the benefits of their applications. Three databases were consulted for eligible papers. The inclusion criteria were in vitro and in vivo preclinical studies, using OA cells or OA animal models, and testing hydrogels and nanoparticles (NPs) over the last ten years. Data extraction and quality assessment were performed. Eleven papers were included. In vitro studies evidenced that NP-gels do not impact on cell viability and do not cause inflammation in OA cell phenotypes. In vivo research on rodents showed that these treatments could increase drug retention in joints, reducing inflammation and preventing articular cartilage damage. Nanotechnologies in preclinical efficacy tests are still new and require extensive studies and technical hits to determine the efficacy, safety, fate, and localization of NPs for translation into an effective therapy for OA patients.

Osteoporosis Preclinical Research: A Systematic Review on Comparative Studies Using Ovariectomized Sheep.

Sheep ovariectomy (OVX) alone or associated to steroid therapy, deficient diet, or hypothalamic-pituitary disconnection has proven to be of critical importance for osteoporosis research in orthopedics. However, the impact of specific variables, such as breed, age, diet, time after OVX, and other variables, should be monitored. Thus, the design of comparative studies is mandatory to minimize the impact of these variables or to recognize the presence of unwanted variables as well as to better characterize bone remodeling in this model. Herein, we conducted a systematic review of the last 10 years on PubMed, Scopus, and Web of Knowledge considering only studies on OVX sheep where a control group was present. Of the 123 records screened, 18 studies were included and analyzed. Results showed that (i) Merino sheep are the most exploited breed; (ii) 5-6 years of age is the most used time for inducing OVX; (iii) ventral midline laparotomy is the most common approach to induce OVX; (iv) OVX associated to steroid therapy is the most widely used osteoporosis model; and (v) success of OVX was mostly verified 12 months after surgery. In detail, starting from 12 months after OVX a significant decline in bone mineral density and in microarchitectural bone parameters as well as in biochemical markers were detected in all studies in comparison to control groups. Bone alteration was also site-specific on a pattern as follows: lumbar vertebra, femoral neck, and ribs. Before 12 months from OVX and starting from 3-5 months, microarchitectural bone changes and biochemical marker alterations were present when osteoporosis was induced by OVX associated to steroid therapy. In conclusion, OVX in sheep influence bone metabolism causing pronounced systemic bone loss and structural deterioration comparable to the situation found in osteoporosis patients. Data for treating osteoporosis patients are based not only on good planning and study design but also on a correct animal use that, as suggested by 3Rs principles and by ARRIVE guidelines, includes the use of control groups to be directly contrasted with the experimental group.

Assessment of the in vivo biofunctionality of a biomimetic hybrid scaffold for osteochondral tissue regeneration.

Chondral and osteochondral lesions represent one of the most challenging problems in the orthopedic field, as these types of injuries lead to disability and worsened quality of life for patients and have an economic impact on the healthcare system. The aim of this in vivo study was to develop a new tissue engineering approach through a hybrid scaffold for osteochondral tissue regeneration made of porous polyurethane foam (PU) coated under vacuum with calcium phosphates (PU/VAC). Scaffold characterization showed a highly porous and interconnected structure. Human amniotic mesenchymal stromal cells (hAMSCs) were loaded into scaffolds using pectin (PECT) as a carrier. Osteochondral defects in medial femoral condyles of rabbits were created and randomly allocated in one of the following groups: plain scaffold (PU/VAC), scaffold with hAMSCs injected in the implant site (PU/VAC/hAMSC), scaffold with hAMSCs loaded in pectin (PU/VAC/PECT/hAMSC), and no treated defects (untreated). The therapeutic efficacy was assessed by macroscopic, histological, histomorphometric, microtomographic, and ultrastructural analyses at 3, 6, 12, and 24 weeks. Histological results showed that the scaffold was permissive to tissue growth and penetration, an immature osteocartilaginous tissue was observed at early experimental times, with a more accentuated bone regeneration in comparison with the cartilage layer in the absence of any inflammatory reaction.

Evaluation of cartilage biomechanics and knee joint microenvironment after different cell-based treatments in a sheep model of early osteoarthritis.

PURPOSE: Aiming to prevent cartilage damage during early osteoarthritis (OA), the therapeutic challenge is to restore and maintain the physiological and functional properties of such a tissue with minimally invasive therapeutic strategies. METHODS: Accordingly, an in vivo model of early OA in sheep was here treated through three different cell therapies (culture expanded ADSCs, SVF, and culture expanded AECs) thus to preserve the joint surface from the progression of the pathology. Three months after the treatment injections, their performance was assessed through mechanical automated mapping (Young's modulus and cartilage thickness), gross evaluation of articular surfaces, and biochemical analysis of the synovial fluid. RESULTS: No severe degeneration was observed after three months from OA induction. Cartilage mechanical properties were crucial to identify early degeneration. All the treatments improved the macroscopic cartilage surface aspect and reduced pro-inflammatory cytokines in the synovial fluid. Among the three treatments, SVF highlighted the best performance while ADSCs the worst. CONCLUSION: Despite that the evaluated experimental time is an early follow-up and, thus, longer trial is mandatory to properly assess treatments effectiveness, the proposed multidisciplinary approach allowed to obtain preliminary, but also crucial, results concerning the reduction in OA signs on cartilage properties, in osteophyte development and in all the inflammatory markers.

Vascular Supply and Bone Marrow Concentrate for the Improvement of Allograft in Bone Defects: A Comparative In Vivo Study.

BACKGROUND: Surgical repair of critical-sized bone defects still remains a big challenge in orthopedic surgery. Biological enhancement, such as growth factors or cells, can stimulate a better outcome in bone regeneration driven by well-established treatments such as allogenic bone graft. However, despite the surgical options available, correct healing can be slowed down or compromised by insufficient vascular supply to the injured site. MATERIALS AND METHODS: In this pilot study, critical size bone defects in rabbit radius were treated with allograft bone, in combination with vascular bundle and autologous bone marrow concentrate seeded onto a commercial collagen scaffold. Microtomographical, histological and immunohistochemical assessments were performed to evaluate allograft integration and bone regeneration. RESULTS: Results showed that the surgical deviation of vascular bundle in the bone graft, regardless from the addition of bone marrow concentrate, promote the onset of healing process at short experimental times (8 wk) in comparison with the other groups, enhancing graft integration. CONCLUSION: The surgical procedure tested stimulates bone healing at early times, preserving native bone architecture, and can be easily combined with biological adjuvant.

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.

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.

Vitamin C: should daily administration keep the paediatrician away?

Vitamin C is an organic compound that is almost ubiquitous in the daily diet of individuals. There are clear indications of supplementation when secondary deficiency is detected related with reduced dietary intake or reduced absorption. On the other hand, indications for supplementation concerning an increased need are controversial. Several authors have studied the role of vitamin C as an adjuvant in the treatment of diseases that may affect children and adolescents. These diseases affect all organs and systems: specifically, vitamin C supplementation could play a role in respiratory, neurological, psychiatric, oncohematological, nephrological, ophthalmological and nutritional disorders. In paediatric age, a significant benefit of vitamin C supplementation has been observed in depressive pathology, iron-deficiency anaemia and chronic renal failure related to haemodialysis. No evidence was found with vitamin C supplementation on mortality, cognitive performance, quality of life, eye diseases, infections, cardiovascular diseases and tumours. This evidence may be related to the fact that in developed countries, vitamin C is almost ubiquitous in the daily diet of each individual. In conclusion, studies on non-industrialised populations in which there could be a real benefit from such supplementation, have yet to be conducted.

Bone marrow concentrate and expanded mesenchymal stromal cell surnatants as cell-free approaches for the treatment of osteochondral defects in a preclinical animal model.

PURPOSE: To evaluate the regenerative potential of surnatants (SNs) from bone marrow concentrate (SN-BMC) and expanded mesenchymal stromal cells (SN-MSCs) loaded onto a collagen scaffold (SC) in comparison with cell-based treatments (BMC and MSCs) in an osteochondral (OC) defect model in rabbits. METHODS: OC defects (3 × 5 mm) were created in the rabbit femoral condyles and treated with SC alone or combined with SN-BMC, SN-MSCs, BMC, and MSCs. In control groups, the defects were left untreated. At three and six months, the quality of regenerated tissue was evaluated with macroscopic, histologic, microtomographic, and immunohistochemical assessments. The production of several immunoenzymatic markers was measured in the synovial fluid. RESULTS: All proposed treatments improved OC regeneration in comparison with untreated and SC-treated defects. Both BMC and MSCs showed a similar healing potential than their respective SNs, with the best performance exerted by BMC as demonstrated with macroscopic and histological scores and type I and II collagen results. CONCLUSIONS: SNs loaded onto SC exerted a positive effect on OC defect regeneration, underlying the biological significance of the trophic factors, thus potentially opening new opportunities for the use of cell-free-based therapies. BMC was confirmed to be the most beneficial treatment.

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.

Effects of pulsed electromagnetic fields and platelet rich plasma in preventing osteoclastogenesis in an in vitro model of osteolysis.

Osteolysis is the main limiting cause for the survival of an orthopedic prosthesis and is accompanied by an enhancement in osteoclastogenesis and inflammation, due by wear debris formation. Unfortunately therapeutic treatments, besides revision surgery, are not available. The aim of the present study was to evaluate the effects of Pulsed Electro Magnetic Fields (PEMFs) and platelet rich plasma (PRP), alone or in combination, in an in vitro model of osteolysis. Rats peripheral blood mononuclear cells were cultured on Ultra High Molecular Weight Polyethylene particles and divided into four groups of treatments: (1) PEMF stimulation (12 hr/day, 2.5 mT, 75 Hz, 1.3 ms pulse duration); (2) 10% PRP; (3) combination of PEMFs, and PRP; (4) no treatment. Treatments were performed for 3 days and cell viability, osteoclast number, expression of genes related to osteoclastogenesis and inflammation and production of pro-inflammatory cytokines were assessed up to 14 days. PEMF stimulation exerted best results because it increased cell viability at early time points and counteracted osteoclastogenesis at 14 days. On the contrary, PRP increased osteoclastogenesis and reduced cell viability in comparison to PEMFs alone. The combination of PEMFs and PRP increased cell viability over time and reduced osteoclastogenesis in comparison to PRP alone. However, these positive results did not exceed the level achieved by PEMF alone. At longer time points PEMF could not counteract osteoclastogenesis increased by PRP. Regarding inflammation, all treatments maintained the production of pro-inflammatory cytokines at low level, although PRP increased the level of interleukin 1 beta.

Polydeoxyribonucleotides (PDRNs) From Skin to Musculoskeletal Tissue Regeneration via Adenosine A2A Receptor Involvement.

Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production, and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo, and clinical studies (1990-2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area. A total of 29 studies were found by PubMed and www.webofknowledge.com searches: 20 were on skin (six in vitro, six in vivo, one vitro/vivo, seven clinical studies), while the other nine regarded bone (one in vitro, two in vivo, one clinical studies), cartilage (one in vitro, one vitro/vivo, two clinical studies), or tendon (one clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity, and reduced pain and inflammation, through the activation of adenosine A2A receptor. PDRNs are currently used for bone, cartilage, and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of three to a maximum of five times and they act trough the activation of A2A receptor. Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. J. Cell. Physiol. 232: 2299-2307, 2017. © 2016 Wiley Periodicals, Inc.

Novel therapeutic targets in osteoarthritis: Narrative review on knock-out genes involved in disease development in mouse animal models.

Osteoarthritis (OA) can affect every joint, especially the knee. Given the complexity of this pathology, OA is difficult to treat with current therapies, which only relieve pain and inflammation and are not capable of restoring tissues once OA has started. Currently, researchers focus on finding a therapeutic strategy that may help to arrest disease progression. The present narrative review gives an overview of the genes involved in the development and progression of OA, assessing in vivo studies performed in knock-out mice affected by OA, to suggest new therapeutic strategies. The article search was performed on the PubMed database and www.webofknowledge.com website with the following keywords: "knee osteoarthritis" AND "knockout mice". The included studies were in English and published from 2005 to 2015. Additional papers were found within the references of the selected articles. In the 55 analyzed in vivo studies, genes mainly affected chondrocyte homeostasis, inflammatory processes, extracellular matrix and the relationship between obesity and OA. Genes are defined as inducing, preventing and not influencing OA. This review shows that joint homeostasis depends on a variety of genetic factors, and preventing or restoring the loss of a gene encoding for protective proteins, or inhibiting the expression of proteins that induce OA, might be a potential therapeutic approach. However, conclusions cannot be drawn because of the wide variability concerning the technique used for OA induction, the role of the genes, the method for tissue evaluations and the lack of assessments of all joint tissues.

Pulsed electromagnetic fields combined with a collagenous scaffold and bone marrow concentrate enhance osteochondral regeneration: an in vivo study.

BACKGROUND: The study aimed to evaluate the combined effect of Pulsed Electromagnetic Field (PEMF) biophysical stimulation and bone marrow concentrate (BMC) in osteochondral defect healing in comparison to the treatment with scaffold alone. METHODS: An osteochondral lesion of both knees was performed in ten rabbits. One was treated with a collagen scaffold alone and the other with scaffold seeded with BMC. Half of the animals were stimulated by PEMFs (75 Hz, 1.5 mT, 4 h/day) and at 40 d, macroscopic, histological and histomorphometric analyses were performed to evaluate osteochondral defect regeneration. RESULTS: Regarding cartilage, the addition of BMC to the scaffold improved cell parameters and the PEMF stimulation improved both cell and matrix parameters compared with scaffold alone. The combination of BMC and PEMFs further improved osteochondral regeneration: there was an improvement in macroscopic, cartilage cellularity and matrix parameters and a reduction in the percentage of cartilage under the tidemark. Epiphyseal bone healing improved in all the osteochondral defects regardless of treatment, although PEMFs alone did not significantly improve the reconstruction of subchondral bone in comparison to treatment with scaffold alone. CONCLUSIONS: Results show that BMC and PEMFs might have a separate effect on osteochondral regeneration, but it seems that they have a greater effect when used together. Biophysical stimulation is a non-invasive therapy, free from side effects and should be started soon after BMC transplantation to increase the quality of the regenerated tissue. However, because this is the first explorative study on the combination of a biological and a biophysical treatment for osteochondral regeneration, future preclinical and clinical research should be focused on this topic to explore mechanisms of action and the correct clinical translation.

Feasibility and safety of electrochemotherapy (ECT) in the pancreas: a pre-clinical investigation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease generally refractory to standard chemotherapeutic agents; therefore improvements in anticancer therapies are mandatory. A major determinant of therapeutic resistance in PDAC is the poor drug delivery to neoplastic cells, mainly due to an extensive fibrotic reaction. Electroporation can be used in vivo to increase cancer cells' local uptake of chemotherapeutics (electrochemotherapy, ECT), thus leading to an enhanced tumour response rate. In the present study, we evaluated the in vivo effects of reversible electroporation in normal pancreas in a rabbit experimental model. We also tested the effect of electroporation on pancreatic cancer cell lines in order to evaluate their increased sensitivity to chemotherapeutic agents. MATERIALS AND METHODS: The application in vivo of the European Standard Operating Procedure of Electrochemotherapy (ESOPE) pulse protocol (1000 V/cm, 8 pulses, 100 µs, 5 KHz) was tested on the pancreas of normal New Zealand White Rabbits and short and long-term toxicity were assessed. PANC1 and MiaPaCa2 cell lines were tested for in vitro electrochemotherapy experiments with and without electroporation. Levels of cell permeabilization were determined by flow cytometry, whereas cell viability and drug (cisplatin and bleomycin) sensitivity of pulsed cells were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: In healthy rabbits, neither systemic nor local toxic effects due to the electroporation procedure were observed, demonstrating the safety of the optimized electric parameters in the treatment of the pancreas in vivo. In parallel, we established an optimized protocol for ECT in vitro that determined an enhanced anti-cancer effect of bleomycin and cisplatin with respect to treatment without electroporation. CONCLUSIONS: Our data suggest that electroporation is a safe procedure in the treatment of PDAC because it does not affect normal pancreatic parenchyma, but has a potentiating effect on cytotoxicity of bleomycin in pancreatic tumour cell lines. Therefore, ECT could be considered as a valid alternative for the local control of non-resectable pancreatic cancer.

Preliminary Evaluation of Bioactive Collagen-Polyphenol Surface Nanolayers on Titanium Implants: An X-ray Photoelectron Spectroscopy and Bone Implant Study.

To endow an implant surface with enhanced properties to ensure an appropriate seal with the host tissue for inflammation/infection resistance, next-generation bone implant collagen-polyphenol nanolayers were built on conventional titanium surfaces through a multilayer approach. X-ray Photoelectron Spectroscopy (XPS) analysis was performed to investigate the chemical arrangement of molecules within the surface layer and to provide an estimate of their thickness. A short-term (2 and 4 weeks) in vivo test of bone implants in a healthy rabbit model was performed to check possible side effects of the soft surface layer on early phases of osteointegration, leading to secondary stability. Results show the building up of the different nanolayers on top of titanium, resulting in a final composite collagen-polyphenol surface and a layer thickness of about 10 nm. In vivo tests performed on machined and state-of-the-art microrough titanium implants do not show significant differences between coated and uncoated samples, as the surface microroughness remains the main driver of bone-to-implant contact. These results confirm that the surface nanolayer does not interfere with the onset and progression of implant osteointegration and prompt the green light for specific investigations of the potential merits of this bioactive coating as an enhancer of the device/tissue seal.