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.

A Pilot Study on Circulating, Cellular, and Tissue Biomarkers in Osteosarcopenic Patients.

Aging comes with the loss of muscle and bone mass, leading to a condition known as osteosarcopenia. Circulating, cellular, and tissue biomarkers research for osteosarcopenia is relatively scarce and, currently, no established biomarkers exist. Here we find that osteosarcopenic patients exhibited elevated basophils and TNFα levels, along with decreased aPPT, PT/INR, IL15, alpha-Klotho, DHEA-S, and FGF-2 expression and distinctive bone and muscle tissue micro-architecture and biomarker expressions. They also displayed an increase in osteoclast precursors with a concomitant imbalance towards spontaneous osteoclastogenesis. Similarities were noted with osteopenic and sarcopenic patients, including a lower neutrophil percentage and altered cytokine expression. A linear discriminant analysis (LDA) on models based on selected biomarkers showed a classification accuracy in the range of 61-78%. Collectively, our data provide compelling evidence for novel biomarkers for osteosarcopenia that may hold potential as diagnostic tools to promote healthy aging.

In Vitro Models of Cell Senescence: A Systematic Review on Musculoskeletal Tissues and Cells.

Ageing is an irreversible and inevitable biological process and a significant risk factor for the development of various diseases, also affecting the musculoskeletal system, resulting from the accumulation of cell senescence. The aim of this systematic review was to collect the in vitro studies conducted over the past decade in which cell senescence was induced through various methods, with the purpose of evaluating the molecular and cellular mechanisms underlying senescence and to identify treatments capable of delaying senescence. Through three electronic databases, 22 in vitro studies were identified and included in this systematic review. Disc, cartilage, or muscle cells or tissues and mesenchymal stem cells were employed to set-up in vitro models of senescence. The most common technique used to induce cell senescence was the addition to the culture medium of tumor necrosis factor (TNF)α and/or interleukin (IL)1ß, followed by irradiation, compression, hydrogen peroxide (H2O2), microgravity, in vitro expansion up to passage 10, and cells harvested from damaged areas of explants. Few studies evaluated possible treatments to anti-senescence effects. The included studies used in vitro models of senescence in musculoskeletal tissues, providing powerful tools to evaluate age-related changes and pathologies, also contributing to the development of new therapeutic approaches.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

Gender-Specific Differences in Human Vertebral Bone Marrow Clot.

Recently, our group described the application of vertebral bone marrow (vBMA) clot as a cell therapy strategy for spinal fusion. Its beneficial effects were confirmed in aging-associated processes, but the influence of gender is unknown. In this study, we compared the biological properties of vBMA clots and derived vertebral mesenchymal stem cells (MSCs) from female and male patients undergoing spinal fusion procedures and treated with vBMA clot. We analyzed the expression of growth factors (GFs) in vBMA clots and MSCs as well as morphology, viability, doubling time, markers expression, clonogenicity, differentiation ability, senescence factors, Klotho expression, and HOX and TALE gene profiles from female and male donors. Our findings indicate that vBMA clots and derived MSCs from males had higher expression of GFs and greater osteogenic and chondrogenic potential compared to female patients. Additionally, vBMA-clot-derived MSCs from female and male donors exhibited distinct levels of HOX and TALE gene expression. Specifically, HOXA1, HOXB8, HOXD9, HOXA11, and PBX1 genes were upregulated in MSCs derived from clotted vBMA from male donors. These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials.

The use of cell conditioned medium for musculoskeletal tissue regeneration.

Tissue regenerative medicine combines the use of cells, scaffolds, and molecules to repair damaged tissues. Different cell types are employed for musculoskeletal diseases, both differentiated and mesenchymal stromal cells (MSCs). In recent years, the hypothesis that cell-based therapy is guided principally by cell-secreted factors has become increasingly popular. The aim of the present literature review was to evaluate preclinical and clinical studies that used conditioned medium (CM), rich in cell-factors, for musculoskeletal regeneration. Thirty-one were in vitro, 12 in vivo studies, 1 was a clinical study, and 2 regarded extracellular vesicles. Both differentiated cells and MSCs produce CM that induces reduction in inflammation and increases synthetic activity. MSC recruitment and differentiation, endothelial cell recruitment and angiogenesis have also been observed. In vivo studies were performed with CM in bone and periodontal defects, arthritis and muscle dystrophy pathologies. The only clinical study was performed with CM from MSCs in patients needing alveolar bone regeneration, showing bone formation and no systemic or local complications. Platelet derived growth factor receptor ß, C3a, vascular endothelial growth factor, monocyte chemoattractant protein-1 and -3, interleukin 3 and 6, insulin-like growth factor-I were identified as responsible of cell migration, proliferation, osteogenic differentiation, and angiogenesis. The use of CM could represent a new regenerative treatment in several musculoskeletal pathologies because it overcomes problems associated with the use of cells and avoids the use of exogenous GFs or gene delivery systems. However, some issues remain to be clarified.

A Human 3D In Vitro Model to Assess the Relationship Between Osteoporosis and Dissemination to Bone of Breast Cancer Tumor Cells.

Despite consistent improvements in diagnostic and therapeutic strategies for breast cancer, up to 40% of patients will develop bone metastases. To reduce the morbidity and complications related with bone metastases, it is imperative to reduce their etiological factors. Osteoporosis, being characterized by a sudden estrogen deficiency, may provide a favorable condition for bone metastasis. This work, using a humanized 3D in vitro model, aims at evaluating the relationship between osteoporosis and breast cancer-derived bone metastases. Bone tissue discarded from total hip replacement surgery of healthy and osteoporotic patients was cultured in a rolling apparatus system in hypoxic environment. Protein levels (i.e., vascular endothelial growth factor (VEGF), VEGF receptor 1, VEGF receptor 2, interleukin (IL)-6, IL-1ß, IL-8 IL-10, tumor necrosis factor α (TNF-α), osteoprotegerin (OPG), receptor activator for nuclear factor KB ligand (RANKL)) and histological and immunohistochemical (i.e., cytokeratin 8 and 18) analyses showed a noticeable specificity of breast cancer cells for the colonization of osteoporotic bone. These data are the first to demonstrate that using humanized 3D in vitro systems, which individually model the pre- and postmenopausal bone microenvironment, it is possible to recognize major differences in tumor growth and colonization between healthy and osteoporotic status. Thus, this system might help to develop a shared system between basic and clinical sciences where a personalized diagnosis is associated to a therapeutic strategy designed for a single patient: a model able to achieve a translational research approach in the clinical setting, which may lead to the application and dissemination of personalized medicine. J. Cell. Physiol. 232: 1826-1834, 2017. © 2016 Wiley Periodicals, Inc.

Increased Chondrogenic Potential of Mesenchymal Cells From Adipose Tissue Versus Bone Marrow-Derived Cells in Osteoarthritic In Vitro Models.

Primarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1ß as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment. 3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1ß, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition, and organization. Both hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1ß by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs. hADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. J. Cell. Physiol. 232: 1478-1488, 2017. © 2016 Wiley Periodicals, Inc.

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.

Multimorbidity and Polytherapy in Patients with Femoral Neck Fracture: A Retrospective Observational Study.

Fractures of the femoral neck are one of the most common reasons for admission to an orthopedic institute. These patients also show multimorbidity (≥2 chronic conditions) and polytherapy (≥5 drugs). Multimorbidity and polytherapy are associated with a high risk of hospitalization and a reduction in quality of life. The present retrospective observational study was conducted to evaluate the prevalence of multimorbidity and polytherapy in patients aged ≥65 years and surgically treated for femoral neck fractures at an orthopedic institute over 3 years. Multimorbidity was evaluated with Elixhauser's comorbidity measure and polytherapy was obtained from the patient's medical record. This study identified 917 patients (84 ± 7.6 years); most of them were females. Most patients presented ≥2 chronic conditions, the most frequent of which was uncomplicated hypertension, and most patients used ≥5 drugs, of which antithrombotic ones were the most frequently taken. No significant gender and age differences were found between the presence or not of multimorbidity or polytherapy. Multimorbidity and polytherapy were statistically associated with an increased and decreased risk of 1-year mortality, respectively. This retrospective study has evaluated the variables required for the establishment of a minimum core of descriptors of the prevalence of polytherapy and multimorbidity in the orthopedic field.

Postoperative Survival and Clinical Outcomes for Uterine Leiomyosarcoma Spinal Bone Metastasis: A Case Series and Systematic Literature Review.

Spinal bone metastases from uterine leiomyosarcoma (LMS) are relatively uncommon and few data are present in the literature. In this study, cases of nine consecutive patients who underwent spinal surgery for metastatic uterine LMS between 2012 and 2022 at a single institution were retrospectively reviewed. The recorded demographic, operative, and postoperative factors were reviewed, and the functional outcomes were determined by changes in Frankel grade classification during follow-up. A systematic review of the literature was also performed to evaluate operative and postoperative factors and outcomes for patients with the same gynecological metastases to the spine. For our cases, the mean time between primary tumors to bone metastases diagnosis was 5.2 years, and the thoracic vertebrae were the most affected segment. Overall, median survival after diagnosis of metastatic spine lesions was 46 months. For the systematic review, the mean time between primary tumors to bone metastases was 4.9 years, with the lumbar spine as the most involved site of metastasis. Overall, median survival after diagnosis was 102 months. Once a spinal bone lesion from LMS is identified, surgical treatment can be beneficial and successful in alleviating symptoms. Further efforts will be crucial to identify prognostic markers as well as therapeutic targets to improve survival in these patients.

Ageing and Osteoarthritis Synergically Affect Human Synoviocyte Cells: An In Vitro Study on Sex Differences.

Osteoarthritis is a chronic inflammatory disease that affects all of the joints, especially those of the elderly. Aging is a natural and irreversible biological process implicated in the pathophysiology of many chronic diseases, such as osteoarthritis. Inflammation and oxidative stress are the main factors involved in osteoarthritis and aging, respectively, with the production of several pro-inflammatory cytokines such as Interleukin 1ß (IL1ß) and reactive oxygen species. The aim of the study was to set-up an in vitro model of osteoarthritis and aging, focusing on the sex differences by culturing male and female fibroblast-like synoviocytes (FLSs) with IL1ß, hydrogen peroxide (H2O2), IL1ß+H2O2 or a growth medium (control). IL1ß+H2O2 reduced the cell viability and microwound healing potential, increased Caspase-3 expression and reactive oxygen species and IL6 production; IL1ß increased IL6 production more than the other conditions did; H2O2 increased Caspase-3 expression and reactive oxygen species production; Klotho expression showed no differences among the treatments. The FLSs from female donors demonstrated a better response capacity in unfavorable conditions of inflammation and oxidative stress than those from the male donors did. This study developed culture conditions to mimic the aging and osteoarthritis microenvironment to evaluate the behavior of the FLSs which play a fundamental role in joint homeostasis, focusing on the sex-related aspects that are relevant in the osteoarthritis pathophysiology.

Sex and gender determinants following spinal fusion surgery: A systematic review of clinical data.

In the last decade, numerous studies analyzed and described the surgical outcomes in male and female patients submitted to orthopedic surgery. Although this, the impact of sex/gender on spinal fusion surgery clinical outcomes is still poorly defined. This review systematically maps and synthesizes the scientific literature on sex/gender differences in postoperative outcomes for patients undergoing spinal fusion surgery. The search was performed in PubMed, Scopus, and Web of Science in the last 22 years. Clinical studies evaluating potential sex/gender differences in postoperative outcomes and/or complications, as primary or secondary aim, were included and analyzed. Out of the 1,885 records screened, 47 studies were included. These studies comprised a total of 1,158,555 patients (51.31% female; 48.69% male). About 77% of the analyzed studies reported sex/gender-related differences in postoperative outcomes. Most studies treated patients for lumbar degenerative diseases and more than 55% of them reported a worse postoperative outcome in female patients in terms of pain, disability, health-related quality of life questionnaires, and complications. Differently, a significant heterogeneity across studies on patients treated for cervical and sacral degenerative diseases as well as for spinal deformity and traumatic spinal fracture prevented the understanding of specific sex/gender differences after spinal fusion surgery. Despite this, the present review highlighted those female patients treated for lumbar degenerative spine diseases could require more clinical awareness during postoperative care. The understanding of how sex/gender differences can really affect clinical outcomes after spinal fusion surgeries is mandatory for all spinal pathological conditions to drive clinical research toward oriented and personalized protocols.

Clinical and Pathological Profiles of Vertebral Bone Metastases from Endometrial Cancers: Evidence from a Twenty-Year Case Series.

Patients with endometrial cancer (EC) frequently have metastases to lungs, extra-pelvic nodes, and liver. Although an uncommon occurrence, cases of EC metastasis to bone, prevalently in vertebral bone, have also been reported. The objective of this study was to analyze clinical and pathological profiles of patients with EC metastatic to vertebral bone. We carried out a retrospective case series on surgically treated patients for this pathology. From 2001 to 2021, out of 775 patients with bone metastasis, 1.6% had bone metastasis from EC. The median time between the diagnosis of primary tumor and that of bone metastases was 31.5 months. Solitary bone lesion was present in 7 patients and lumbar vertebrae were the segments most affected. Pathological fractures in 46.2% of patients and spinal pain in all were present. In terms of location, 46.2% of bone metastases resided within the anterior section of the vertebra, while the remaining presented an extension within the anterior and posterior sections, with 46.1% of cases showing an extradural extra-osseous extension and paraspinous envelope. Median survival after diagnosis of bone metastasis was 11.5 months. Vertebral bone metastasis in EC is a rare phenomenon, with severe prognosis. An in-depth understanding of this topic may guide future management and treatment decisions, thus improving life expectancy and quality.

Mesenchymal stem cells in the aging and osteoporotic population.

Because of their ability to self-renew and differentiate, mesenchymal stem cells (MSCs) are the in vivo source for replacing lost cells in high-turnover tissues during the life of an organism. MSCs have osteogenic potential and can be eligible for the repair and maintenance of the skeleton, thus they are very attractive for tissue engineering and regenerative medicine approaches. However, many changes in their behavior, caused by aging and bone disease, have been reported in the literature. These changes, which affect MSC self-renewal ability and differentiation potentiality, are related to cell proliferation, differentiation, cell cycle phases (depending on gene modification), and cytokine and growth factor production. This review summarizes the literature related to intrinsic and extrinsic characteristics of human bone marrow or adipose tissue MSCs during aging and osteoporosis. Although some studies reveal contrasting results, the results of this review suggest that the cellular modifications due to aging and osteoporosis should be carefully considered in relation to the use of MSCs for therapeutic application.

In vivo preclinical evaluation of the influence of osteoporosis on the anchorage of different pedicle screw designs.

We investigate the anchorage of pedicle screws with different surface treatments in osteoporotic bone. Eight ewes were divided into two groups of four animals each: four sheep underwent bilateral ovariectomy (OVX Group), whereas the operation was simulated in the remaining group (SHAM Group). Eighteen months after the first operation, the Dynesys(®) System was fitted to the sheep using pedicle screws with three different surface treatments: untreated, rough blasted (uncoated) and bioactive coated (bioactive). Uncoated screws showed a significantly higher bone ingrowth value compared with the untreated screws in the OVX group (9.3%, p < 0.005) and a significantly lower bone ingrowth value in the SHAM group (-11.0%, p < 0.05). Furthermore, the bioactive pedicle screws had a significant lower bone ingrowth value than the untreated screws in the SHAM group (-12.1%, p < 0.05). These results suggest that both tested surface treatments of pedicular screws may provide an advantage in terms of bone quality and osseointegration, when implanted in osteoporotic vertebrae.

Association of Klotho with physical performance and frailty in middle-aged and older adults: A systematic review.

Ageing is an inevitable process of physical deterioration that impairs functional autonomy and quality of life, becoming a public health issue. Since the percentage of people over 60 years is increasing worldwide, the use of easily detectable biomarkers of ageing is a relevant tool for monitoring of the ageing process and treatment. Among them, Klotho, an ageing suppressor gene because its deficiency leads to ageing like phenotype, seems particularly promising. This systematic review includes the last 10 years clinical studies that evaluated the association between plasma Klotho and body composition, physical performance and frailty in both sedentary and active middle-aged and older adults. Sixteen studies have been found: nine regarding the association between Klotho and body composition, two the association of Klotho and frailty and finally five concerning the effects of physical activity on Klotho. The results of these studies, albeit with some exceptions, point out that Klotho is positively associated with muscle strength and negatively with osteoporosis, frailty, disability and mortality while physical activity generally increases Klotho levels. Moreover, even if there are still few clinical studies, Klotho might be positively associated with bone mineral density, muscle strength, longevity, mobility and robustness during ageing.

Gender and Sex Are Key Determinants in Osteoarthritis Not Only Confounding Variables. A Systematic Review of Clinical Data.

Many risk factors for osteoarthritis (OA) have been noted, while gender/sex differences have been understated. The work aimed to systematically review literature investigating as primary aim the relationship between gender/sex related discriminants and OA. The search was performed in PubMed, Science Direct and Web of Knowledge in the last 10 years. Inclusion criteria were limited to clinical studies of patients affected by OA in any joints, analyzing as primary aim gender/sex differences. Exclusion criteria were review articles, in vitro, in vivo and ex vivo studies, case series studies and papers in which gender/sex differences were adjusted as confounding variable. Of the 120 records screened, 42 studies were included. Different clinical outcomes were analyzed: morphometric differences, followed by kinematics, pain, functional outcomes after arthroplasty and health care needs of patients. Women appear to use more health care, have higher OA prevalence, clinical pain and inflammation, decreased cartilage volume, physical difficulty, and smaller joint parameters and dimensions, as compared to men. No in-depth studies or mechanistic studies analyzing biomarker differential expressions, molecular pathways and omic profiles were found that might drive preclinical and clinical research towards sex-/gender-oriented protocols.

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.