Histological and histomorphometric analysis of bone tissue using customized titanium meshes with or without resorbable membranes: A randomized clinical trial.

OBJECTIVES: To date, no clinical studies have investigated the effect of using resorbable collagen membrane in conjunction with customized titanium mesh to promote bone formation in guided bone regeneration. Therefore, a non-inferiority analysis (one-sided 95% CI approach) was designed to compare the augmented bone gained using meshes with and without collagen membranes, through histological and histomorphometric investigations. MATERIALS AND METHODS: Thirty patients undergoing bone augmentation procedures at both maxillary and mandible sites were randomly treated with customized titanium meshes alone (M-, n = 15) or covered with resorbable membrane (M+, n = 15), in both cases filled with autogenous bone and xenograft. After 6 months of healing, bone tissue biopsies were taken from the augmented region. The bone tissue (B.Ar), grafting material (G.Ar), and non-mineralized tissue (NMT.Ar) areas were quantified through histomorphometric analysis, as were the osteoid area (O.Ar) and its width. RESULTS: Collagen membrane did not appear to significantly influence the investigated parameters: B.Ar, G.Ar, NMT.Ar, and O.Ar were similar between Group M- (34.3%, 11.5%, 54.1%, 1.95 µm2 , respectively) and Group M+ (35.3%, 14.6%, 50.2%, and 1.75 µm2 , respectively). Considering the overall population, significantly higher rates of newly formed bone were obtained in mandibular sites, while non-mineralized and dense connective tissue rates were higher in the maxilla (p < .05). CONCLUSIONS: The application of collagen membrane over titanium mesh did not lead to significant results. Bone formation appeared significantly different in the maxilla compared with the mandible. Additional studies are required to further investigate the issues observed.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

RNA Extraction from Cartilage: Issues, Methods, Tips.

The increase in degenerative diseases involving articular cartilage has pushed research to focus on their pathogenesis and treatment, exploiting increasingly complex techniques. Gene expression analyses from tissue are representative of the in vivo situation, but the protocols to be applied to obtain a reliable analysis are not completely cleared through customs. Thus, RNA extraction from fresh samples and specifically from musculoskeletal tissue such as cartilage is still a challenging issue. The aim of the review is to provide an overview of the techniques described in the literature for RNA extraction, highlighting limits and possibilities. The research retrieved 65 papers suitable for the purposes. The results highlighted the great difficulty in comparing the different studies, both for the sources of tissue used and for the techniques employed, as well as the details about protocols. Few papers compared different RNA extraction methods or homogenization techniques; the case study reported by authors about RNA extraction from sheep cartilage has not found an analog in the literature, confirming the existence of a relevant blank on studies about RNA extraction from cartilage tissue. However, the state of the art depicted can be used as a starting point to improve and expand studies on this topic.

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.

Vitamin D and Platelets: A Menacing Duo in COVID-19 and Potential Relation to Bone Remodeling.

Global data correlate severe vitamin D deficiency with COVID-19-associated coagulopathy, further suggesting the presence of a hypercoagulable state in severe COVID-19 patients, which could promote thrombosis in the lungs and in other organs. The feedback loop between COVID-19-associated coagulopathy and vitamin D also involves platelets (PLTs), since vitamin D deficiency stimulates PLT activation and aggregation and increases fibrinolysis and thrombosis. Vitamin D and PLTs share and play specific roles not only in coagulation and thrombosis but also during inflammation, endothelial dysfunction, and immune response. Additionally, another 'fil rouge' between vitamin D and PLTs is represented by their role in mineral metabolism and bone health, since vitamin D deficiency, low PLT count, and altered PLT-related parameters are linked to abnormal bone remodeling in certain pathological conditions, such as osteoporosis (OP). Hence, it is possible to speculate that severe COVID-19 patients are characterized by the presence of several predisposing factors to bone fragility and OP that may be monitored to avoid potential complications. Here, we hypothesize different pervasive actions of vitamin D and PLT association in COVID-19, also allowing for potential preliminary information on bone health status during COVID-19 infection.

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.

Platelet functions and activities as potential hematologic parameters related to Coronavirus Disease 2019 (Covid-19).

Coronavirus disease 2019 (COVID-19) is a new infectious disease that currently lacks standardized and established laboratory markers to evaluate its severity. In COVID-19 patients, the number of platelets (PLTs) and dynamic changes of PLT-related parameters are currently a concern. The present paper discusses the potential link between PLT parameters and COVID-19. Several studies have identified a link between severe COVID-19 patients and specific coagulation index, in particular, high D-dimer level, prolonged prothrombin time, and low PLT count. These alterations reflect the hypercoagulable state present in severe COVID-19 patients, which could promote microthrombosis in the lungs, as well as in other organs. Further information and more advanced hematological parameters related to PLTs are needed to better estimate this link, also considering COVID-19 patients at different disease stages and stratified in different cohorts based on preexisting co-morbidity, age, and gender. Increasing the understanding of PLT functions in COVID-19 will undoubtedly improve our knowledge on disease pathogenesis, clinical management, and therapeutic options, but could also lead to the development of more precise therapeutic strategies for COVID-19 patients.

Platelet Features and Derivatives in Osteoporosis: A Rational and Systematic Review on the Best Evidence.

Background: With the increase in aging population, the rising prevalence of osteoporosis (OP) has become an important medical issue. Accumulating evidence showed a close relationship between OP and hematopoiesis and emerging proofs revealed that platelets (PLTs), unique blood elements, rich in growth factors (GFs), play a critical role in bone remodeling. The aim of this review was to evaluate how PLT features, size, volume, bioactive GFs released, existing GFs in PLTs and PLT derivatives change and behave during OP. Methods: A systematic search was carried out in PubMed, Scopus, Web of Science Core Collection and Cochrane Central Register of Controlled Trials databases to identify preclinical and clinical studies in the last 10 years on PLT function/features and growth factor in PLTs and on PLT derivatives during OP. The methodological quality of included studies was assessed by QUIPS tool for assessing risk of bias in the clinical studies and by the SYRCLE tool for assessing risk of bias in animal studies. Results: In the initial search, 2761 studies were obtained, only 47 articles were submitted to complete reading, and 23 articles were selected for the analysis, 13 on PLT function/features and growth factor in PLTs and 10 on PLT derivatives. Risk of bias of almost all animal studies was high, while the in the clinical studies risk of bias was prevalently moderate/low for the most of the studies. The majority of the evaluated studies highlighted a positive correlation between PLT size/volume and bone mineralization and an improvement in bone regeneration ability by using PLTs bioactive GFs and PLT derivatives. Conclusions: The application of PLT features as OP markers and of PLT-derived compounds as therapeutic approach to promote bone healing during OP need to be further confirmed to provide clear evidence for the real efficacy of these interventions and to contribute to the clinical translation.

Osteochondral tissue cultures: Between limits and sparks, the next step for advanced in vitro models.

The increasing demand for reliable preclinical models and to reduce, refine and, if possible, replace animal studies have brought forth the development of complex tissue cultures in different research areas, including the musculoskeletal field. In this paper, we review the literature within last 10 years on the state of progress for in vitro models of osteochondral tissue cultures, taking into account the clinical relevance of the management and treatment of osteochondral lesions. According to the selected research criteria, 35 works, 27 of which with animal tissues and 8 with human tissues, resulted to be relevant for the purposes of this review. Data analyzed revealed a great heterogeneity among the proposed tissue culture models. The anatomical harvesting sites resulted to be mainly the knee stifle joint, both for animal (prevalently bovines) and human tissues derived from joint replacement surgery, and significant heterogeneity among culture conditions and media were found. To date, very few papers have focused on the set up of a reproducible in vitro model, applicable to a variety of studies, thus suggesting a relevant gap to fill in the development of advanced three-dimensional osteochondral culture models.

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.

Peripheral Blood Mononuclear Cells Spontaneous Osteoclastogenesis: Mechanisms Driving the Process and Clinical Relevance in Skeletal Disease.

In vitro peripheral blood mononuclear cells differentiate into osteoclasts under the influence of osteoclast-stimulating factors. However, accumulating evidence suggests spontaneous osteoclasts formation and activity in patients affected by local or systemic bone remodeling diseases in comparison with healthy controls. Therefore, within this review, we summarize the studies where spontaneous osteoclastogenesis of peripheral blood mononuclear cells was observed in pathological conditions of the skeletal system. We indicate a linkage between immunoregulation by T cells and spontaneous osteoclasts formation with increased levels of tumor necrosis factors-α, receptor activator of nuclear factor kappa-B ligand and inteleukin-7 production. In the light of these results, it would be of crucial importance to deepen the correlation between systemic bone remodeling diseases and spontaneous osteoclastogenesis as well as to investigate in detail the mechanisms underlying this phenomenon and the clinical relevance in bone remodeling disease diagnosis and monitoring.

Vitamin D Level Between Calcium-Phosphorus Homeostasis and Immune System: New Perspective in Osteoporosis.

Vitamin D is a key molecule in calcium and phosphate homeostasis; however, increasing evidence has recently shown that it also plays a crucial role in the immune system, both innate and adaptive. A deregulation of vitamin D levels, due also to mutations and polymorphisms in the genes of the vitamin D pathway, determines severe alterations in the homeostasis of the organism, resulting in a higher risk of onset of some diseases, including osteoporosis. This review gives an overview of the influence of vitamin D levels on the pathogenesis of osteoporosis, between bone homeostasis and immune system.

Ionized jet deposition of silver nanostructured coatings: Assessment of chemico-physical and biological behavior for application in orthopedics.

Infection is one of the main issues connected to implantation of biomedical devices and represents a very difficult issue to tackle, for clinicians and for patients. This study aimed at tackling infection through antibacterial nanostructured silver coatings manufactured by Ionized Jet Deposition (IJD) for application as new and advanced coating systems for medical devices. Films composition and morphology depending on deposition parameters were investigated and their performances evaluated by correlating these properties with the antibacterial and antibiofilm efficacy of the coatings, against Escherichia coli and Staphylococcus aureus strains and with their cytotoxicity towards human cell line fibroblasts. The biocompatibility of the coatings, the nanotoxicity, and the safety of the proposed approach were evaluated, for the first time, in vitro and in vivo by rat subcutaneous implant models. Different deposition times, corresponding to different thicknesses, were selected and compared. All silver coatings exhibited a highly homogeneous surface composed of nanosized spherical aggregates. All coatings having a thickness of 50 nm and above showed high antibacterial efficacy, while none of the tested options caused cytotoxicity when tested in vitro. Indeed, silver films impacted on bacterial strains viability and capability to adhere to the substrate, in a thickness-dependent manner. The nanostructure obtained by IJD permitted to mitigate the toxicity of silver, conferring strong antibacterial and anti-adhesive features, without affecting the coatings biocompatibility. At the explant, the coatings were still present although they showed signs of progressive dissolution, compatible with the release of silver, but no cracking, delamination or in vivo toxicity was observed.

In vivo validation of highly customized cranial Ti-6AL-4V ELI prostheses fabricated through incremental forming and superplastic forming: an ovine model study.

Cranial reconstructions are essential for restoring both function and aesthetics in patients with craniofacial deformities or traumatic injuries. Titanium prostheses have gained popularity due to their biocompatibility, strength, and corrosion resistance. The use of Superplastic Forming (SPF) and Single Point Incremental Forming (SPIF) techniques to create titanium prostheses, specifically designed for cranial reconstructions was investigated in an ovine model through microtomographic and histomorphometric analyses. The results obtained from the explanted specimens revealed significant variations in bone volume, trabecular thickness, spacing, and number across different regions of interest (VOIs or ROIs). Those regions next to the center of the cranial defect exhibited the most immature bone, characterized by higher porosity, decreased trabecular thickness, and wider trabecular spacing. Dynamic histomorphometry demonstrated differences in the mineralizing surface to bone surface ratio (MS/BS) and mineral apposition rate (MAR) depending on the timing of fluorochrome administration. A layer of connective tissue separated the prosthesis and the bone tissue. Overall, the study provided validation for the use of cranial prostheses made using SPF and SPIF techniques, offering insights into the processes of bone formation and remodeling in the implanted ovine model.

A Systematic Review of the Retrograde Drilling Approach for Osteochondral Lesion of the Talus: Questioning Surgical Approaches, Outcome Evaluation and Gender-Related Differences.

BACKGROUND: Retrograde drilling (RD) is a minimally invasive surgical procedure mainly used for non-displaced osteochondral lesions (OCL) of the talus, dealing with subchondral necrotic sclerotic lesions or subchondral cysts without inducing iatrogenic articular cartilage injury, allowing the revascularization of the subchondral bone and new bone formation. METHODS: This systematic review collected and analyzed the clinical studies of the last 10 years of literature, focusing not only on the clinical results but also on patients' related factors (gender, BMI, age and complications). RESULTS: Sixteen clinical studies were retrieved, and differences in the type of study, follow-up, number and age of patients, lesion type, dimensions, grades and comparison groups were observed, making it difficult to draw conclusions. Nevertheless, lesions on which RD showed the best results were those of I-III grades and not exceeding 150 mm2 in size, showing overall positive results, a good rate of patient satisfaction, improvements in clinical scores, pain reduction and return to daily activities and sports. CONCLUSIONS: There are still few studies dealing with the issue of post-surgical complications and gender-related responses. Further clinical or preclinical studies are thus mandatory to underline the success of this technique, also in light of gender differences.

Blood factors as biomarkers in osteoporosis: points from the COVID-19 era.

The restrictions adopted during the coronavirus disease 2019 (COVID-19) pandemic limiting direct medical consultations and access to healthcare centers reduced the participation of patients with chronic diseases, such as osteoporosis (OP), in screening and monitoring programs. This highlighted the need for new screening diagnostic tools that are clinically effective, but require minimal technical and time commitments, to stratify populations and identify who is more at risk for OP and related complications. This paper provides an overview of the potential use of blood-related factors, such as platelet (PLT)- and monocyte-related factors, as biomarkers able to quickly screen, detect, and monitor OP in both sexes. Such biomarkers might be of key importance not only during the COVID-19 pandemic but also, even more importantly, during periods of better global health stability.

Skin adhesion to the percutaneous component of direct bone anchored systems: systematic review on preclinical approaches and biomaterials.

Nowadays, direct bone anchored systems are an increasingly adopted approach in the therapeutic landscape for amputee patients. However, the percutaneous nature of these devices poses a major challenge to obtain a stable and lasting proper adhesion between the implant surface and the skin. A systematic review was carried out in three databases (PubMed, Scopus, Web of Science) to provide an overview of the innovative strategies tested with preclinical models (in vitro and in vivo) in the last ten years to improve the skin adhesion of direct bone anchored systems. Fifty five articles were selected after screening, also employing PECO question and inclusion criteria. A modified Cochrane RoB 2.0 tool for the in vitro studies and the SYRCLE tool for in in vivo studies were used to assess the risk of bias. The evidence collected suggests that the implementation of porous percutaneous structures could be one of the most favorable approach to improve proper skin adhesion, especially in association with bioactive coatings, as hydroxyapatite, and exploiting the field of nanostructure. Some issues still remain open as (a) the identification and characterization of the best material/coating association able to limit the shear stresses at the interface and (b) the role of keratinocyte turnover on the skin/biomaterial adhesion and integration processes.

Determination of the Spatial Anisotropy of the Surface MicroStructures of Different Implant Materials: An Atomic Force Microscopy Study.

Many biomaterials' surfaces exhibit directional properties, i.e., possess spatial anisotropy on a range of spatial scales spanning from the domain of the naked eye to the sub-micrometer level. Spatial anisotropy of surface can influence the mechanical, physicochemical, and morphological characteristics of the biomaterial, thus affecting its functional behavior in relation, for example, to the host tissue response in regenerative processes, or to the efficacy of spatially organized surface patterns in avoiding bacterial attachment. Despite the importance of the availability of quantitative data, a comprehensive characterization of anisotropic topographies is generally a hard task due to the proliferation of parameters and inherent formal complications. This fact has led so far to excessive simplification that has often prevented researchers from having comparable results. In an attempt to overcome these issues, in this work a systematic and multiscale approach to spatial anisotropy is adopted, based on the determination of only two statistical parameters of surface, namely the texture aspect ratio Str and the roughness exponent H, extracted from atomic force microscopy images of the surface. The validity on this approach is tested on four commercially available implant materials, namely titanium alloy, polyethylene, polyetheretherketone and polyurethane, characterized by textured surfaces obtained after different machining. It is found that the "two parameters" approach is effective in describing the anisotropy changes on surfaces with complex morphology, providing a simple quantitative route for characterization and design of natural and artificial textured surfaces at spatial scales relevant to a wide range of bio-oriented applications.

Stromal Vascular Fraction and Amniotic Epithelial Cells: Preclinical and Clinical Relevance in Musculoskeletal Regenerative Medicine.

Musculoskeletal regenerative medicine is mainly based on the use of cell therapy to heal damaged tissues such as bone, cartilage, and tendons. Throughout the years, different cell types have been employed for the treatment of musculoskeletal diseases, in particular, mesenchymal stem cells (MSCs) derived from bone marrow (BMSCs) and adipose tissue (ADSCs). Though the results of these literature studies have been encouraging, there are some limitations, especially on long-term results. Recently, some interest has shifted towards new cell types such as the stromal vascular fraction (SVF) and amniotic endothelial cells (AECs). The aim of the present literature review is to evaluate preclinical and clinical studies that used SVF and AECs for musculoskeletal tissue regeneration. Forty-eight preclinical and clinical studies, performed in the last 10 years, were identified. Both SVF and AECs, injected or implanted with or without scaffolds, were shown to be valid alternatives, and in some ways superior, to ADSCs and BMSCs, being able to differentiate towards osteogenic, chondrogenic, and tenogenic lineages, and to promote cell and tissue regenerative potential. The use of SVF and AECs could represent a new regenerative treatment in several musculoskeletal pathologies, solving the problem of cell expansion in vitro.