Impact of COVID-19 Pandemic on Treatment and Outcome of Fragility Hip Fractures In Non-COVID Patients: Comparison Between the Lockdown Period, a Historical Series and the "Pandemic Normality" in a Single Institution.

Introduction: The COVID-19 pandemic has affected and is still deeply affecting all aspects of public life. World governments have been forced to enact restrictive measures to stem the contagion which have led to a decrease in the movement of people within national territory and to a redirection of health care resources with a suspension of non-urgent procedures. In Italy, a lockdown was imposed from March 9th to May 3rd, 2020. As a result, a significant reduction in the overall operative volume of orthopedic trauma was expected, but it was not possible to predict a similar trend regarding fragility fractures of the proximal femur in the elderly. Methods: The aim of this paper was to examine the impact of COVID-19 on the operating volume for trauma surgeries and to determine how the pandemic affected the management of fragility hip fractures (FHFs) in non-COVID patients at a single Institution. Results: The first result was a statistically significant reduction in the overall operative volume of orthopedic trauma during the period of the first lockdown and an increase in the mean age of patients undergoing surgery, as expected. As regard to the second aim, the incidence of FHFs remained almost unchanged during the periods analysed. The population examined were superimposable in terms of demographics, comorbidities, type of fracture, peri-operative complications, percentage of operations performed within 48 hours from hospitalization and 1-year outcome. Discussion: Our results are in line with those already present in the Literature. Conclusions: Our study revealed a significant impact of the restrictive anti-contagion measures on the overall orthopedic surgical volume, but, at the same time, we could affirm that the pandemic did not affect the management of FHFs in non-COVID patients, and their results.

Treatment Algorithm of Periprosthetic Femoral Fracturens.

Introduction. The ever-expanding indications for total hip arthroplasty are leading to more implants being placed in younger as well as in older patients with high functional demand. Also, prolonged life expectancy is contributing to an overall increment of periprosthetic femoral fractures. The Vancouver classification has been the most used for guiding the surgeon choice since its proposal in 1995. Fractures occurring over a hip femoral implant can be divided into intra-operative and post-operative PFFs, and their treatment depends on factors that may severely affect the outcome: level of fracture, implant stability, quality of bone stock, patients' functional demand, age and comorbidities, and surgeon expertise. There are many different treatment techniques available which include osteosynthesis and revision surgery or a combination of both. The goals of surgical treatment are patients' early mobilization, restoration of anatomical alignment and length with a stable prosthesis and maintenance of bone stock. Significance. The aim of this review is to describe the state-of-the-art treatment and outcomes in the management of PFFs. We performed a systematic literature review of studies reporting on the management of PFFs around hip stems and inter-prosthetic fractures identifying 45 manuscripts eligible for the analysis. Conclusions. PFFs present peculiar characteristic that must be considered and special features that must be addressed. Their management is complex due to the extreme variability of stem designs, the possibility of having cemented or uncemented stems, the difficulty in identifying the "real" level of the fracture and the actual stability of the stem. As a result, the definition of a standardized treatment is unlikely, thereby high expertise is fundamental for the surgical management of PPFs, so this kind of fractures should be treated only in specialized centres with both high volume of revision joint arthroplasty and trauma surgery.

Surgical and Pharmacological Management of Periprosthetic Atypical Femoral Fractures: A Narrative Literature Review.

Introduction: An increasing number of patients is annually undergoing total hip arthroplasty (THA), and a significant proportion of these patients are elderly and consequently at a higher risk of complications because of age, osteoporosis, and medical comorbidities. Periprosthetic femoral fractures (PFFs) are one of the worst complications of THA associated with high rates of unfavorable prognosis. Besides, in the last decade, a new independent disease entity called "atypical femoral fracture" (AFF) has been identified and defined by the American Society for Bone and Mineral Research (ASBMR) task force. Some PFFs present clinical history and radiographic aspect consistent with an AFF, meeting the ASBMR criteria for the diagnosis of AFF except that PFFs by themselves are an exclusion criterion for AFF. However, there is an increasing number of published studies suggesting that periprosthetic atypical femoral fractures (PAFFs) exist and should not be excluded by definition. Significance: Nowadays, although there is an increasing interest in PAFFs, there are still very few studies published on the topic and a lack of consensus regarding their treatment. This narrative literature review aims to introduce this new emerging topic to a wider readership describing the characteristics of PAFFs and the state-of-the-art in their management. Conclusions: Many authors agree that PAFFs should be considered as a subgroup of PFFs that have atypical characteristics; they also show a significant correlation with prolonged bisphosphonate use. A correct diagnosis is paramount for proper treatment of the disease that requires both surgical and medical actions to be taken.

Periprosthetic Atypical Femoral Fractures Exist: A Retrospective Study at a Single Institution. Prevalence on 115 Periprosthetic Femoral Fractures Around a Primary Hip Stem.

BACKGROUND: Some periprosthetic femoral fractures (PFFs) present history and radiographic aspect consistent with an atypical femoral fracture (AFF), fulfilling the criteria for AFF except that PFFs by themselves are excluded from the diagnosis of AFFs. The aim of this study is to evaluate in a single institution series of PFFs if any of them could be considered a periprosthetic atypical femoral fracture (PAFF), and their prevalence. METHODS: Surgical records were searched for PFFs around a primary hip stem from January 2013 to December 2019. Cases were classified according to Vancouver classification. Demographic and medical history was extracted. Fisher's exact test was used for statistical analysis. RESULTS: One hundred fifteen PFFs were identified, 59 of them were type B1 and 16 were type C. Radiographs and medical records were available for all patients. Twenty-four patients (32%) have been treated with bisphosphonates (BPs) for longer than 4 years. Four patients presented a fracture with characteristics of PAFF. When enlarged to all PFFs of the series, no other PAFF was found: prevalence of PAFFs was 5.3% for type B1 and C cases and 3.5% for all surgically treated PFFs. Statistical significative difference between PAFFs and PFFs was found for prolonged BP assumption and for the level of fracture clear of the stem. CONCLUSION: Fracture with characteristics of AFFs can also happen over a prosthetic stem, configuring themselves as PAFFs, and they are related to prolonged BP use. As a correct diagnosis is mandatory for proper treatment, a revision of criteria for AFFs should be considered, accepting that PAFFs exist.

Combined Surgical and Medical Treatment for Vancouver B1 and C Periprosthetic Femoral Fractures: A Proposal of a Therapeutic Algorithm While Retaining the Original Stable Stem.

INTRODUCTION: There is lack of consensus regarding best operative fixation strategy for periprosthetic femoral fractures (PFFs) around a stable stem. Evidence exists that some patterns of fracture around a stable stem are better treated with revision surgery than with standard fixation. Anyway, a more aggressive surgical procedure together with medical treatment could allow for stem retention, and reduced risk of nonunion/hardware failure, even in these cases. SIGNIFICANCE: This paper is placed in a broader context of lack of studies on the matter, and its aim is to shed some light on the management of PFFs around a stable stem, when peculiar mechanical and biological aspects are present. RESULTS: Based on our casuistry in the treatment of nonunions after PFF successfully treated with original stem retention, and on review of Literature about risk factors for fixation failure, an algorithm is proposed that can guide in choosing the ideal surgical technique even for first-time PFFs with a stable stem, without resorting to revision. Mechanical (major and minor) and biological (local and systemic) factors that may influence fracture healing, leading to nonunion and hardware failure, and subsequent need for re-operation, are considered. The proposed surgical technique consists of rigid fixation with absolute stability (using a plate and structural allograft) plus local biological support (structural allograft and autologous bone marrow concentrate over a platelet-rich plasma-based scaffold) at fracture site. Systemic anabolic treatment (Teriparatide) is also administered in the post-operative period. CONCLUSION: Mechanical factors are not the only issues to be considered when choosing the surgical approach to PFFs over a stable stem. Systemic and local biological conditions should be taken into account, as well. A therapeutic algorithm is proposed, given the prosthetic stem to be stable, considering mechanical and biological criteria.

Medial pivot vs posterior stabilized total knee arthroplasty designs: a gait analysis study.

Aim To compare a medial pivot (MP) total knee arthroplasty (TKA) with posterior stabilized (PS) TKA designs from a subjective, clinical and biomechanical point of view, in a single-centre, single-surgeon, case-control non-randomized trial. Methods Sixteen patients were randomly picked up from case series into each group. Subjective outcome was assessed using the Forgotten Joint Score Questionnaire (FJSQ). Clinical evaluation included range of motion (ROM). All patients underwent gait analysis by a treadmill with force-measuring plaques and videorecording device; data were recorded for 30 seconds and included cadence, step length, stance time and walking speed. A blinded qualitative analysis of the pattern of gait was defined as biphasic or non-biphasic. Descriptive statistics for the continuous study variables and statistical significance were calculated for all parameters with independent-samples t-test and χ2 test to analyse difference in pattern of gait between groups. Results Mean FJSQ in the MP group was 91.87 (CI 95%: 88.12- 95.46) and 75.31 (CI 95%: 67.97-81.56) in the PS group (p=0.029). Mean post-operative ROM was 117° (CI 95%: 113°-122°) in the MP group and 112° (CI 95%: 108°-117°) in the PS group (p=0.14). No statistical difference was found between groups regarding all gait analysis parameters which have been recorded. Conclusion MP TKA design showed better subjective results using the FJSQ, but it did not improve significantly clinical and functional outcomes compared to PS TKA design, at a short-term follow-up.

LARS versus hamstring tendon autograft in anterior cruciate ligament reconstruction: a single-centre, single surgeon retrospective study with 8 years of follow-up.

PURPOSE: The choice of graft type in the anterior cruciate ligament (ACL) reconstruction remains a subject of controversy. The aim of this study was to assess the outcomes in ACL reconstructions performed using a four-strand hamstring tendon graft (4SHG) or a LARS ligament comparing the effectiveness of the two grafts at a medium follow-up of 8 years. METHODS: This retrospective, single-centre, single surgeon study evaluated the clinical, functional and radiographic outcomes in 50 patients who underwent ACL reconstruction (25 4SHG and 25 LARS). Patients who underwent surgery after more than 6 months from injury and showed radiographically visible degenerative changes at time of surgery were excluded from the study. RESULTS: None of the patients underwent re-surgery in the same knee. The range of motion of the operated knee, compared to the contralateral, was good for both groups. The anterior drawer test resulted negative in 21 patients (84%) in the LARS group and eight patients (32%) in the 4SHG group (P = 0.039). The Lachman test was negative in 19 patients (76%) in the LARS group and in 11 patients (44%) in the 4SHG group (P = 0.045). Although other results of ACL reconstruction measured by Lysholm scores, IKDC evaluation, Tegner scores and radiographic images showed using a LARS graft tended to be superior to using a 4SHG, there were no statistically significant differences calculated. CONCLUSION: Our results suggest that 4 years after ACL reconstruction using a LARS ligament or 4SHG dramatically improves the function outcome, while the patients in the LARS group displayed a higher knee stability than those in the 4SHG group.

T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset.

Novel nano-composite biomimetic biomaterial allows chondrogenic and osteogenic differentiation of bone marrow concentrate derived cells.

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of osteochondral lesions. For this purpose, the ideal scaffold should possess the right properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, mechanical strength, and flexibility. In addition, it should be easy to handle and serve as a template for chondrocyte and bone cells guiding both cartilage and bone formation. The aim of the present study was to estimate the chondrogenic and osteogenic capability of bone marrow concentrated derived cells seeded onto a novel nano-composite biomimetic material. These properties have been evaluated by means of histological, immunohistochemical and electron microscopy analyses. The data obtained demonstrated that freshly harvested cells obtained from bone marrow were able, once seeded onto the biomaterial, to differentiate either down the chondrogenic and osteogenic pathways as evaluated by the expression and production of specific matrix molecules. These findings support the use, for the repair of osteochondral lesions, of this new nano-composite biomimetic material together with bone marrow derived cells in a "one step" transplantation procedure.

Effect of two different preparations of platelet-rich plasma on synoviocytes.

PURPOSE: To analyse the modifications induced by two different platelet-rich plasma (PRP) preparations on osteoarthritis (OA) synoviocytes, by documenting changes in gene expression of factors involved in joint physiopathology. METHODS: OA synoviocytes were cultured for 7 days in medium with different concentrations of either P-PRP (a pure platelet concentrate without leucocytes but with a limited number of platelets), L-PRP (a higher platelet concentrate containing leucocytes) or platelet-poor plasma (PPP). Gene expression of interleukin (IL)-1beta, IL-6, IL-8/CXCL8, tumour necrosis factor alpha, IL-10, IL-4, IL-13, metalloproteinase-13, tissue inhibitor of metalloproteinase (TIMP)-1, (TIMP)-3, (TIMP)-4, vascular endothelial growth factor, transforming growth factor beta1, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), hyaluronic acid (HA) synthases (HAS)-1, (HAS)-2, and (HAS)-3 was analysed by RT-PCR. HA production was determined in culture supernatants by ELISA. RESULTS: IL-1ß, IL-8 and FGF-2 were significantly induced by L-PRP compared to both P-PRP and PPP; HGF was down-modulated by L-PRP versus both P-PRP and PPP, and an inverse dose-response influence was shown for all preparations. Expression level of TIMP-4 was lower in the presence of L-PRP compared with P-PRP. HA production and HAS gene expression did not seem to be modulated by PRP. CONCLUSIONS: L-PRP is able to sustain the up-regulation of proinflammatory factors, (IL-1beta, IL-8 and FGF-2), together with a down-modulation of HGF and TIMP-4 expression, two factors that have been recognized as anti-catabolic mediators in cartilage, thus supporting the need to further optimize the PRP preparations to be applied in clinical practice.

Hydroxytyrosol prevents increase of osteoarthritis markers in human chondrocytes treated with hydrogen peroxide or growth-related oncogene α.

Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene α (GROα), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GROα. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GROα. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features.

Does platelet-rich plasma freeze-thawing influence growth factor release and their effects on chondrocytes and synoviocytes?

PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1ß, HGF, PDGF AB/BB, TGF-ß1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-ß1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1ß and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

Platelet-rich plasma affects bacterial growth in vitro.

BACKGROUND AIMS: Platelet-rich plasma (PRP), a blood derivative rich in platelets, is a relatively new technique used in tissue regeneration and engineering. The increased quantity of platelets makes this formulation of considerable value for their role in tissue healing and microbicidal activity. This activity was investigated against five of the most important strains involved in nosocomial infections (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus faecalis) to understand the prophylactic role of pure (P)-PRP. Microbicidal proteins released from activated P-PRP platelets were also determined. METHODS: The microbicidal activity of P-PRP and platelet-poor plasma (PPP) was evaluated on different concentrations of the five bacterial strains incubated for 1, 2, 4 and 18 h and plated on agar for 18-24 h. P-PRP and PPP-released microbicidal proteins were evaluated by means of multiplex bead-based immunoassays. RESULTS: P-PRP and PPP inhibited bacterial growth for up to 2 h of incubation. The effect of P-PRP was significantly higher than that of PPP, mainly at the low seeding concentrations and/or shorter incubation times, depending on the bacterial strain. Chemokine (C-C motif) ligand-3, chemokine (C-C motif) ligand-5 and chemokine (C-X-C motif) ligand-1 were the molecules mostly related to Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis inhibition. Escherichia coli and Klebsiella pneumoniae were less influenced. CONCLUSIONS: The present results show that P-PRP might supply an early protection against bacterial contaminations during surgical interventions because the inhibitory activity is already evident from the first hour of treatment, which suggests that physiological molecules supplied in loco might be important in the time frame needed for the activation of the innate immune response.

Sodium hydrosulfide inhibits the differentiation of osteoclast progenitor cells via NRF2-dependent mechanism.

Hydrogen sulfide (H2S), which recently emerged as a potent regulator of tissues and organs, is broadly produced in mammalian cells but whether it can regulate bone cell function is still elusive. The main objective of this study was to establish the role of H2S in the regulation of human osteoclast differentiation and function. Sodium hydrosulfide (NaHS), a common H2S-donor, was administered in vitro to CD11b+ human monocytes, the pool of circulating osteoclasts precursors which are critically involved in osteoclast development and function in bone. NaHS dose-dependently decreased human osteoclast differentiation at concentrations which did not induce toxicity. The inhibition of human osteoclast differentiation was associated with a down-regulation in RANKL-dependent intracellular ROS levels in human pre-osteoclasts cells. Furthermore, NaHS up-regulated NRF2 protein expression, its nuclear translocation, and the transcription of the two key downstream antioxidant genes Peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1, suggesting that NRF2 activation may inhibit human osteoclast differentiation by activating a sustained antioxidant response in osteoclast progenitors; furthermore, NRF2 activators Sulforaphane and Tert-butylhydroquinone inhibited in vitro human osteoclast differentiation. Moreover, silencing NRF2 in human pre-osteoclasts totally abolished NaHS-mediated inhibition of osteoclastogenesis, suggesting that NRF2 is essential to the inhibitory function of NaHS in osteoclast development. Finally, we found that NaHS also downregulated the RANKL/OPG mRNA ratio in human mesenchymal stem cells, the key osteoclast-supporting cells. Our results suggest that NaHS shows a potential therapeutical role in erosive diseases of bone by regulating both direct and indirect mechanisms controlling the differentiation of circulating osteoclasts precursors.

Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1ß-stimulated production of chemokines and matrix-degrading enzymes in vitro.

BACKGROUND: In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1ß. METHODOLOGY/PRINCIPAL FINDINGS: The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1ß and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1ß-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1ß/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. CONCLUSIONS/SIGNIFICANCE: Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1ß-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

Signaling pathways in cartilage repair.

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair.

Chondrogenic potential of Slug-depleted human mesenchymal stem cells.

The use of short interfering RNA (siRNA) in combination with stem cells and biocompatible scaffolds is a promising strategy in regenerative medicine. Our experimental strategy was to explore the possibility of forcing or guiding the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) by knocking down a negative regulator of chondrogenesis, Slug transcription factor (TF), thus altering cell behavior. We found that TGFß-driven chondrogenic differentiation of hMSCs cultured onto a hyaluronan-based scaffold, HYAFF(®)-11, was strengthened after cell exposure to siRNA against Slug. Slug silencing was effective in promoting the expression of chondrogenic markers, including Col2A1, aggrecan, Sox9, LEF1, and TRPS1. In addition, we confirmed that HYAFF-11 is a good scaffold candidate for hMSC use in tissue engineering applications, and showed that it is effective in sustaining TGFß3 treatment associated with a specific gene silencing. Interestingly, preliminary results from the experimental model described here suggested that, even in the absence of differentiation supplements, Slug silencing showed a pro-chondrogenic effect, highlighting both its potential use as an alternative to TGFß treatment, and the critical role of the Slug TF in determining the fate of hMSCs.

Comparison of platelet-rich plasma formulations for cartilage healing: an in vitro study.

BACKGROUND: Platelet-rich plasma (PRP) has been advocated as one treatment for cartilage tissue regeneration. To date, several different platelet-rich formulations have been available, but a deep knowledge of their composition and mechanism of action in a specific clinical use is needed. The aim of this study was to investigate the effect of various PRP formulations on human chondrocytes in vitro. METHODS: Blood from ten human volunteers was used to prepare three formulations: (1) PRP with a relatively low concentration of platelets and very few leukocytes (P-PRP), (2) PRP with high concentrations of both platelets and leukocytes (L-PRP), and (3) platelet-poor plasma (PPP). Selected growth factors in the formulations were measured, and the in vitro effects of various concentrations were tested by exposing chondrocytes isolated from osteoarthritic cartilage of four different men and measuring cell proliferation, matrix production, and gene expression. RESULTS: L-PRP contained the highest levels of growth factors and cytokines. All three formulations stimulated chondrocyte proliferation throughout the culture period evaluated; the only significant difference among the formulations was on day 7, when P-PRP induced greater cell growth compared with the other two formulations. P-PRP stimulated chondrocyte anabolism, as shown by the expression of type-II collagen and aggrecan, whereas L-PRP promoted catabolic pathways involving various cytokines. However, L-PRP induced greater expression of the hyaluronic acid synthase-2 gene and greater production of hyaluronan compared with P-PRP. CONCLUSIONS: L-PRP and P-PRP induced distinct effects on human articular chondrocytes in vitro, possibly because of differences in the concentrations of platelets, leukocytes, growth factors, and other bioactive molecules. The identification of the optimal amounts and ratios of these blood components could ideally lead to a formulation more suitable for the treatment of cartilage lesions.

Polyamine delivery as a tool to modulate stem cell differentiation in skeletal tissue engineering.

The first step in skeleton development is the condensation of mesenchymal precursors followed by any of two different types of ossification, depending on the type of bone segment: in intramembranous ossification, the bone is deposed directly in the mesenchymal anlagen, whereas in endochondral ossification, the bone is deposed onto a template of cartilage that is subsequently substituted by bone. Polyamines and polyamine-related enzymes have been implicated in bone development as global regulators of the transcriptional and translational activity of stem cells and pivotal transcription factors. Therefore, it is tempting to investigate their use as a tool to improve regenerative medicine strategies in orthopedics. Growing evidence in vitro suggests a role for polyamines in enhancing differentiation in both adult stem cells and differentiated chondrocytes. Adipose-derived stem cells have recently proved to be a convenient alternative to bone marrow stromal cells, due to their easy accessibility and the high frequency of stem cell precursors per volume unit. State-of-the-art "prolotherapy" approaches for skeleton regeneration include the use of adipose-derived stem cells and platelet concentrates, such as platelet-rich plasma (PRP). Besides several growth factors, PRP also contains polyamines in the micromolar range, which may also exert an anti-apoptotic effect, thus helping to explain the efficacy of PRP in enhancing osteogenesis in vitro and in vivo. On the other hand, spermidine and spermine are both able to enhance hypertrophy and terminal differentiation of chondrocytes and therefore appear to be inducers of endochondral ossification. Finally, the peculiar activity of spermidine as an inducer of autophagy suggests the possibility of exploiting its use to enhance this cytoprotective mechanism to counteract the degenerative changes underlying either the aging or degenerative diseases that affect bone or cartilage.

Human adipose stromal cells (ASC) for the regeneration of injured cartilage display genetic stability after in vitro culture expansion.

Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.