Arianna B. Lovati

Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells

The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Bone marrow, umbilical cord and amniotic fluid samples were harvested from different horses. Cells were analyzed for proliferation, immunocytochemical, stem cell gene expression and multilineage plasticity. BM- and AF-MSCs took similar time to reach confluence and showed comparable plating efficiency. All cell lines expressed identical stem cell markers and capability to differentiate towards osteogenic lineage. Almost all cell lines differentiated into the adipogenic lineage as demonstrated by cytochemical staining, even if no adipose gene expression was detectable for AF-MSCs. AF- and EUC-MSCs showed a limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. These findings suggest that AF-MSCs appeared to be a readily obtainable and highly proliferative cell line from an uninvasive source that may represent a good model system for stem cell biology. More studies are needed to investigate their multilineage potential. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation.

Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review

Tendon ruptures are a great burden in clinics. Finding a proper graft material as a substitute for tendon repair is one of the main challenges in orthopaedics, for which the requirement of a biological scaffold would be different for each clinical application. Among biological scaffolds, the use of decellularized tendon-derived matrix increasingly represents an interesting approach to treat tendon ruptures. We analyzed in vitro and in vivo studies focused on the development of efficient protocols for the decellularization and for the cell reseeding of the tendon matrix to obtain medical devices for tendon substitution. Our review considered also the proper tendon source and preclinical animal models with the aim of entering into clinical trials. The results highlight a wide panorama in terms of allogenic or xenogeneic tendon sources, specimen dimensions, physical or chemical decellularization techniques, and the cell type variety for reseeding from terminally differentiated to undifferentiated mesenchymal stem cells and their static or dynamic culture employed to generate implantable constructs tested in different animal models. We try to identify the most efficient approach to achieve an optimal biological scaffold for biomechanics and intrinsic properties, resembling the native tendon and being applicable in clinics in the near future, with particular attention to the Achilles tendon substitution.

Diabetic Mouse Model of Orthopaedic Implant-Related Staphylococcus Aureus Infection

Background Periprosthetic bacterial infections represent one of the most challenging orthopaedic complications that often require implant removal and surgical debridement and carry high social and economical costs. Diabetes is one of the most relevant risk factors of implant-related infection and its clinical occurrence is growing worldwide. The aim of the present study was to test a model of implant-related infection in the diabetic mouse, with a view to allow further investigation on the relative efficacy of prevention and treatment options in diabetic and non-diabetic individuals. Methodology A cohort of diabetic NOD/ShiLtJ mice was compared with non-diabetic CD1 mice as an in vivo model of S. aureus orthopaedic infection of bone and soft tissues after femur intramedullary pin implantation. We tested control and infected groups with 1×103 colony-forming units of S. aureus ATCC 25923 strain injected in the implant site. At 4 weeks post-inoculation, host response to infection, microbial biofilm formation, and bone damage were assessed by traditional diagnostic parameters (bacterial culture, C-reactive protein and white blood cell count), histological analysis and imaging techniques (micro computed tomography and scanning electron microscopy). Results Unlike the controls and the CD1 mice, all the diabetic mice challenged with a single inoculum of S. aureus displayed severe osteomyelitic changes around the implant. Conclusions Our findings demonstrate for the first time that the diabetic mouse can be successfully used in a model of orthopaedic implant-related infection. Furthermore, the same bacteria inoculum induced periprosthetic infection in all the diabetic mice but not in the controls. This animal model of implant-related infection in diabetes may be a useful tool to test in vivo treatments in diabetic and non-diabetic individuals.

Tenogenic differentiation of equine mesenchymal progenitor cells under indirect co-culture.

PURPOSE Adult bone marrow mesenchymal stem cells (BM-MSCs) are a potential cell source for tendon repair in direct cell therapy and tissue engineering investigations. The purpose of this study was to evaluate the tenogenic induction of undifferentiated BM-MSCs under indirect co-culture technique with trimmed native tendon tissue. Since the horse represents a preferred species to study tendon regenerative strategies, this work was conducted on equine BM-MSCs. METHODS Equine BM-MSCs were co-cultured in a transwell system with tendon tissue fragments. The BM-MSC tenogenic differentiation was evaluated by cytochemical staining and real time PCR for gene expression. Cell viability in tendon fragments and cultured cells was analyzed. RESULTS Our results indicate that under indirect co-culture with native and healthy tendon tissue the BM-MSCs expressed tendon-specific markers such as decorin, tenomodulin, tenascin-C, and collagen type I. They also retained a tenocyte-like phenotype during monolayer culture. CONCLUSIONS Data are very encouraging for future in vitro investigations into committing cells to the tenogenic lineage without adding growth factors or serum to the culture medium for both cell therapy and tissue engineering.

Modeling Staphylococcus epidermidis -Induced Non-Unions: Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (103, 105, 108 colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 103 group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 105 and 108 groups showed severe signs of osteomyelitis and a non-union rate of 83–100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.

Fabrication of multi‐well chips for spheroid cultures and implantable constructs through rapid prototyping techniques

Three‐dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi‐well chips, which were validated using articular chondrocytes (ACs). Multi‐well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type‐II collagen deposition. Moreover, the use of our multi‐well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical‐grade fibrin to generate implantable multi‐well constructs allowing for the precise distribution of multiple cell types. Multi‐well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi‐well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi‐well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre‐implantation samples. In conclusion, multi‐well chips for the generation and culture of multiple cell spheroids can be fabricated by low‐cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment. Biotechnol. Bioeng. 2015;112: 1457–1471.

Different combinations of growth factors for the tenogenic differentiation of bone marrow mesenchymal stem cells in monolayer culture and in fibrin-based three-dimensional constructs

Tendon injuries are severe burdens in clinics. The poor tendon healing is related to an ineffective response of resident cells and inadequate vascularization. Thanks to the high proliferation and multi-lineage differentiation capability, bone marrow-derived mesenchymal stem cells (BMSCs) are a promising cell source to support the tendon repair. To date, the association of various growth factors to induce the in vitro tenogenic differentiation of multipotent progenitor cells is poorly investigated. This study aimed to investigate the tenogenic differentiation of rabbit BMSCs by testing the combination of bone morphogenetic proteins (BMP-12 and 14) with transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) both in 2D and 3D cultures within fibrin-based constructs. After 7 and 14 days, the tenogenic differentiation was assessed by analyzing cell metabolism and collagen content, the gene expression of tenogenic markers and the histological cell distribution and collagen deposition within 3D constructs. Our results demonstrated that the association of BMP-14 with TGF-β3 and VEGF enhanced the BMSC tenogenic differentiation both in 2D and 3D cultures. This study supports the use of fibrin as hydrogel-based matrix to generate spheroids loaded with tenogenic differentiated BMSCs that could be used to treat tendon lesions in the future.

Animal Models of Implant-Related Low-Grade Infections. A Twenty-Year Review.

The demand for joint replacement and surgical treatment is continuously increasing, thus representing a clinical burden and a cost for the healthcare system. Among several pathogens involved in implant-related infections, staphylococci account for the two-thirds of clinically isolated bacteria. Despite most of them are highly virulent microorganisms (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa), low virulent bacteria (Staphylococcus epidermidis, Propionibacterium acnes) are responsible for delayed, low-grade infections without specific clinical signs and hardly distinguishable from aseptic prosthetic failure. Therefore, there is a real need to study the pathogenesis of orthopedic infections through in vivo animal models. The present review of the literature provides a 20-year overview of animal models of acute, subclinical or chronic orthopedic infections according to the pathogen virulence and inocula. Through this analysis, a great variety of conditions in terms of bacterial strains and inocula emerged, thus encouraging the development of more reproducible in vivo studies to provide relevant information for a translational approach to humans.

A review on animal models and treatments for the reconstruction of Achilles and flexor tendons

Tendon is a connective tissue mainly composed of collagen fibers with peculiar mechanical properties essential to functional movements. The increasing incidence of tendon traumatic injuries and ruptures—associated or not with the loss of tissue—falls on the growing interest in the field of tissue engineering and regenerative medicine. The use of animal models is mandatory to deepen the knowledge of the tendon healing response to severe damages or acute transections. Thus, the selection of preclinical models is crucial to ensure a successful translation of effective and safe innovative treatments to the clinical practice. The current review is focused on animal models of tendon ruptures and lacerations or defective injuries with large tissue loss that require surgical approaches or grafting procedures. Data published between 2000 and 2016 were examined. The analyzed articles were compiled from Pub Med-NCBI using search terms, including animal model(s) AND tendon augmentation OR tendon substitute(s) OR tendon substitution OR tendon replacement OR tendon graft(s) OR tendon defect(s) OR tendon rupture(s). This article presents the existing preclinical models – considering their advantages and disadvantages—in which translational progresses have been made by using bioactive sutures or tissue engineering that combines biomaterials with cells and growth factors to efficiently treat transections or large defects of Achilles and flexor tendons.Graphical Abstract

In vivo evaluation of bone deposition in macroporous titanium implants loaded with mesenchymal stem cells and strontium-enriched hydrogel.

Bone-implant integration represents a major requirement to grant implant stability and reduce the risk of implant loosening. This study investigates the effect of progenitor cells and strontium-enriched hydrogel on the osseointegration of titanium implants. To mimic implant-bone interaction, an ectopic model was developed grafting Trabecular Titanium(™) (TT) implants into decellularized bone seeded with human bone marrow mesenchymal stem cells (hBMSCs). TT was loaded or not with strontium-enriched amidated carboxymethylcellulose (CMCA) hydrogel and/or hBMSCs. Constructs were implanted subcutaneously in athymic mice and osteodeposition was investigated with microcomputed tomography (micro-CT), scanning electron microscopy (SEM), and pull-out test at 4, 8, and 12 weeks. Fluorescence imaging was performed at 8 and 12 weeks, histology at 4 and 8 weeks. Micro-CT demonstrated the homogeneity of the engineered bone in all groups, supporting the reproducibility of the ectopic model. Fluorescence imaging, histology, SEM and pull-out mechanical testing showed superior tissue ingrowth in TT implants loaded with both strontium-enriched CMCA and hBMSCs. In our model, the synergic action of the bioactive hydrogel and hBMSCs increased both the bone deposition and TT integration. Thus, we suggest that using orthopedic prosthetic implant preloaded with strontium-enriched CMCA and seeded with BMSCs could represent a valid single-step surgical strategy to improve implant osseointegration.