Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored.
Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration.
Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma.
Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels2 to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis. Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies.
Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers.
Relevant Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages. Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy.
Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra. pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity. We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active. The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies.
It is strongly recommended that tissue and synovial fluid culture samples be obtained during reimplantation performed as part of a two-stage exchange arthroplasty. The incidence of positive cultures during reimplantation and the influence of positive cultures on subsequent outcome are unknown. This aim of this study was to determine the incidence of positive cultures during reimplantation and to investigate the association between positive cultures at reimplantation and the subsequent outcome A retrospective review was conducted on 267 patients that met the Musculoskeletal Infection Society (MSIS) criteria for PJI that completed both stages of two-stage exchange arthroplasty (Table 1). Intraoperative culture results from tissue and/or synovial fluid were obtained. Cultures were positive in 33 cases (12.4%) undergoing reimplantation surgery (Figure 1). Treatment failure was assessed based on the Delphi consensus definition. Logistic regression analysis was performed to assess the predictors of positive culture and risk factors for failure of two-stage exchange arthroplasty. Treatment failure was 45.5% for those with a positive intraoperative culture and 20.9% in those with negative cultures at the time of reimplantation. When controlling for organism virulence, comorbidities, and other confounding factors, treatment failure was higher (odds ratio [OR]: 3.3; 95% confidence interval [CI]: 1.3–4.5) and occurred at an earlier time point (hazard ratio: 2.5; 95% CI: 1.3–4.5) in patients with a positive reimplantation culture. The treatment failure rate was not different between cases with two or more positive cultures (36.4%) and one positive culture (42.8%). Positive intraoperative cultures during reimplantation, regardless of the number of positive samples were independently associated with two times the risk of subsequent infection and earlier treatment failure. Surgeons should be aware that a positive culture at the time of reimplantation independently increases the risk of subsequent failure and needs to be taken seriously. Given the significance of these findings, future studies are needed to evaluate the optimal management of positive cultures during reimplantation surgery.
Preoperative antibiotic prophylaxis remains one of the most important strategies for preventing periprosthetic joint infection (PJI). Current guidelines recommend giving universal antibiotic prophylaxis to all total joint arthroplasty (TJA) patients regardless of their medical conditions or immune status. The aims of this study were to determine if comorbidities influence the organism profile of PJIs and to investigate if the efficacy of the two most frequently used perioperative antibiotics (cefazolin or vancomycin) are affected by patient comorbidities. Using an institutional database, the influence of comorbidities on the organism profile of 1022 PJIs was evaluated. To investigate the influence of perioperative antibiotic monotherapy (cefazolin or vancomycin therapy) on PJI, 8575 primary TJAs were identified and analyzed based on their comorbidities. Patients with multiple perioperative antibiotics, prior septic arthritis, unavailable perioperative antibiotic information, or who underwent aseptic revision were excluded. PJI was determined from ICD-9 codes. While no comorbidities were associated with an increased rate of gram-positive or gram-negative infections, metastatic disease (odds ratio [OR] 7.54, p=0.006), rheumatologic disease (OR 1.63, p=0.046), and chronic pulmonary disease (OR 1.46, p=0.030) demonstrated an increased risk of Staphylococcus aureus PJI. In addition, metastatic disease (OR 5.71, 95% confidence interval [CI] 1.12–26.93, p=0.018), congestive heart failure (OR 2.2, 95% CI 1.16–4.00, p=0.010), chronic pulmonary disease (OR 1.76; 95% CI 1.09–2.78, p=0.015), and diabetes (OR 1.66; 95% CI 1.08–2.52, p=0.019) were associated with PJI from antibiotic resistant organisms. However, there was no difference in the rate of PJI between cefazolin and vancomycin monotherapy when stratified for the aforementioned comorbidities. The present study reveals that comorbidities do not significantly alter the organism profile of high-risk comorbidities and that comorbidities associated with immune deficits do not influence the rate of PJI between two different antibiotics. The results of this study thus support current guidelines, which provide a universal recommendation rather than a protocol that is tailored to a patient's preexisting comorbidities.
Failure of a two-stage exchange arthroplasty for management of periprosthetic joint infection (PJI) poses a major clinical challenge. There is a paucity of information regarding the outcome of further surgical intervention in these patients. Thus, we aim to report the clinical outcomes of subsequent surgical intervention following a failed prior two-stage exchange. Our institutional database was used to identify 60 patients (42 knees and 18 hips) with a failed prior two-stage exchange from infection, who underwent further surgical intervention between 1998 and 2012 and had a minimum of two years follow-up. A retrospective review was performed to extract relevant clinical information, such as mortality, microbiology, and subsequent surgeries. Musculoskeletal Infection Society criteria were used to define PJI, and treatment success was defined using the Delphi criteria as previously reported. Irrigation and debridement (I&D) was performed after a failed two-stage exchange in 61.7% (37/60) patients. The failure rate of I&D in this cohort was 51.3% (19/37). Two patients underwent amputation after I&D due to uncontrolled infection. A total of 40 patients underwent an intended a second two-stage exchange. Reimplantation occurred in only 65% of cases (26/40), and infection was controlled in 61.6% (16/26) of patients. An interim spacer exchange was required in 15% (6/40) of the cases. Of the 14 cases that did not undergo a second stage reimplantation, 5 required amputation, 6 had retained spacers, 1 underwent arthrodesis, and 2 patients died. Further surgical intervention after a failed prior two-stage exchange has poor outcomes. I&D has a high failure rate and many of the patients who are deemed candidates for a second two-stage exchange either do not undergo reimplantation for various reasons or fail after reimplantation. The management of PJI clearly remains imperfect, and there is a dire need for further innovations that may improve the care of these PJI patients.
Introduction: Arthrodesis of the wrist must still be considered as a useful procedure in the treatment of certain deformities of the wrist joint that by performing this operation can improve the function or the aesthetics of the limb. Except those techniques of partial carpal arthrodesis, the surgical procedures of wrist arthrodesis requires a bridging from the radius to the metacarpal in order to stabilize the joint. When this procedure is performed in a growing child this can be a draw back. Material: We have developed a new procedure that producing the arthrodesis distally to the growing cartilage of the radius does not interfere with the growing at wrist level. Furthermore, the use of a wire shroud gives an active fixation reducing postoperative immobilisation and shortening healing time. Since 1986 we have performed this technique in 9 cases of children with mean age of 14 years. The pathology was in 5 cases Cerebral Palsy, in 2 cases Juvenile Rheumatoid Arthritis and in 2 cases Obstetrical Brachial Plexus Palsy. Eight cases were males and 3 cases females. The indication for surgery was flexion deformity of the wrist in 8 cases and extension in 1 case. Four cases had carpal instability (including the 2 Juvenile Rheumatoid Arthritis). Results: The time of fusion was in all cases 2 months with primary arthrodesis and improved extremity. Functional improvement seemed to be most related to pre-operative conditions. Follow up ranged from 4 years to 6 years. Conclusions: The good results obtained with this procedure encourage us to present this new surgical technique to be applied in the still growing child.
