Fetal Immunomodulatory Environment Following Cartilage Injury—The Key to CARTILAGE Regeneration?

Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.     

The Potential of Using an Autogenous Tendon Graft by Injecting Bone Marrow Aspirate in a Rabbit Meniscectomy Model

Bone marrow aspirate (BMA) is an excellent source of cells and growth factors and has been used successfully for bone, cartilage, and soft-tissue healing. This study aimed to investigate the histological and biomechanical properties of autogenous tendon graft by injecting BMA and its protective effect against degenerative changes in a rabbit model of meniscal defects. Adult white rabbits were divided into untreated, tendon, and tendon + BMA groups, and meniscal defects were created in the knees. The tendon graft and articular cartilage status were evaluated by macroscopic and histological analysis at 4, 12, and 24 weeks postoperatively among the three groups. The tendon graft in the tendon and tendon + BMA groups were used for biomechanical evaluation at 4, 12, and 24 weeks postoperatively. The meniscal covering ratio in the tendon + BMA group was better than that in the tendon and untreated groups at 12 and 24 weeks postoperatively. The matrix around the central portion of cells in the tendon + BMA group was positively stained by safranin O and toluidine blue staining with metachromasia at 24 weeks. The histological score of the tendon graft in the tendon + BMA group was significantly higher than that in the untreated and tendon groups at 12 and 24 weeks postoperatively. In the tendon + BMA group, cartilage erosion was not shown at 4 weeks, developed slowly, and was better preserved at 12 and 24 weeks compared to the untreated and tendon groups. Histological scores for the articular cartilage were significantly better in the tendon + BMA group at 24 weeks. The compressive stress on the tendon graft in the tendon + BMA group was significantly higher than that in the tendon group at 12 weeks postoperatively. Transplantation of autogenous tendon grafts by injecting BMA improved the histologic score of the regenerated meniscal tissue and was more effective than the tendon and untreated group for preventing cartilage degeneration in a rabbit model of massive meniscal defects.     

A Tendon-Specific Double Reporter Transgenic Mouse Enables Tracking Cell Lineage and Functions Alteration In Vitro and In Vivo

We generated and characterized a transgenic mouse line with the tendon-specific expression of a double fluorescent reporter system, which will fulfill an unmet need for animal models to support real-time monitoring cell behaviors during tendon development, growth, and repair in vitro and in vivo. The mScarlet red fluorescent protein is driven by the Scleraxis (Scx) promoter to report the cell lineage alteration. The blue fluorescent protein reporter is expressed under the control of the 3.6kb Collagen Type I Alpha 1 Chain (Col1a1) proximal promoter. In this promoter, the existence of two promoter regions named tendon-specific cis-acting elements (TSE1, TSE2) ensure the specific expression of blue fluorescent protein (BFP) in tendon tissue. Collagen I is a crucial marker for tendon regeneration that is a major component of healthy tendons. Thus, the alteration of function during tendon repair can be estimated by BFP expression. After mechanical stimulation, the expression of mScarlet and BFP increased in adipose-derived mesenchymal stem cells (ADMSCs) from our transgenic mouse line, and there was a rising trend on tendon key markers. These results suggest that our tendon-specific double reporter system is a novel model used to study cell re-differentiation and extracellular matrix alteration in vitro and in vivo.     

Therapeutic Effects of Platelet-Derived Extracellular Vesicles in a Bioengineered Tendon Disease Model

Tendon injuries represent over 30–50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restoring. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regeneration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.     

Extracellular Vesicles of Adipose-Derived Stem Cells Promote the Healing of Traumatized Achilles Tendons

Healing of ruptured tendons remains a clinical challenge because of its slow progress and relatively weak mechanical force at an early stage. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have therapeutic potential for tissue regeneration. In this study, we isolated EVs from adipose-derived stem cells (ADSCs) and evaluated their ability to promote tendon regeneration. Our results indicated that ADSC-EVs significantly enhanced the proliferation and migration of tenocytes in vitro. To further study the roles of ADSC-EVs in tendon regeneration, ADSC-EVs were used in Achilles tendon repair in rabbits. The mechanical strength, histology, and protein expression in the injured tendon tissues significantly improved 4 weeks after ADSC-EV treatment. Decorin and biglycan were significantly upregulated in comparison to the untreated controls. In summary, ADSC-EVs stimulated the proliferation and migration of tenocytes and improved the mechanical strength of repaired tendons, suggesting that ADSC-EV treatment is a potential highly potent therapeutic strategy for tendon injuries.     

Bone Regeneration in rat using a gelatin/bioactive glass nanocomposite scaffold along with endothelial cells (HUVECs)

In our previous study, a three‐dimensional gelatin/bioactive glass nanocomposite scaffold with a total porosity of about 85% and pore sizes ranging from 200 to 500 μm was prepared through layer solvent casting combined with lamination technique. The aim of this study was to evaluate in vitro biocompatibility and in vivo bone regeneration potential of these scaffolds with and without endothelial cells when implanted into a critical‐sized rat calvarial defect. MTT assay, SEM observation, and DAPI staining were used to evaluate cell viability and adhesion in macroporous scaffolds and results demonstrated that the scaffolds were biocompatible enough to support cell attachment and proliferation. To investigate the in vivo osteogenesis of the scaffold, blank scaffolds and endothelial/scaffold constructs were implanted in critical‐sized defects, whereas in control group defects were left untreated. Bone regeneration and vascularization were evaluated at 1, 4, and 12 weeks postsurgery by histological, immunohistochemical, and histomorphometric analysis. It was shown that both groups facilitated bone growth into the defect area but improved bone regeneration was seen with the incorporation of endothelial cells. The data showed that the porous Gel/BaG nanocomposite scaffolds could well support new bone formation, indicating that the proposed strategy is a promising alternative for tissue‐engineered bone defects.     

Electrospun Nanofibrous Composite Membranes for Long-Term Release of Celecoxib for Achilles Tendon Reconstruction Following Injury

Embedding multiple pharmaceutical drugs into poly(lactic-co-glycolic acid) (PLGA) nanofibers is known to improve the regeneration of the Achilles tendon upon implantation after injury and rapidly restore post-surgery activity. In this study, an implantable material comprising celecoxib, collagen, bupivacaine, and PLGA (CCBP) is prepared by electrospinning. Its in vitro/vivo drug discharge behaviors are evaluated, and its efficacy in tendon regeneration is investigated in a rat model. The regeneration capacity of the wounded tendon is also compared with that of a doxycycline-collagen-bupivacaine-PLGA (DCBP) combination. The results show that, relative to the primary PLGA nanofibers, the pharmaceutical-embedded nanofibers have thinner fiber diameters and higher hydrophilicity. The drug-eluting nanofibers also offer a sustained release of celecoxib for at least 30 d in vitro and 28 d in vivo. Achilles tendons regenerated using the CCBP combination nanofibers demonstrate a significantly higher maximum load-to-failure than normal tendons and those repaired using the DCBP combination. Additionally, the expression of growth factors and composition of collagen I induced by the CCBP combination are superior to those induced by the DCBP combination. The results suggest that the CCBP combination is a promising scaffold for the repair of ruptured Achilles tendons.     

Multi-Layer Electrospun Membrane Mimicking Tendon Sheath for Prevention of Tendon Adhesions

Defect of the tendon sheath after tendon injury is a main reason for tendon adhesions, but it is a daunting challenge for the biomimetic substitute of the tendon sheath after injury due to its multi-layer membrane-like structure and complex biologic functions. In this study, a multi-layer membrane with celecoxib-loaded poly(l-lactic acid)-polyethylene glycol (PELA) electrospun fibrous membrane as the outer layer, hyaluronic acid (HA) gel as middle layer, and PELA electrospun fibrous membrane as the inner layer was designed. The anti-adhesion efficacy of this multi-layer membrane was compared with a single-layer use in rabbit flexor digitorum profundus tendon model. The surface morphology showed that both PELA fibers and celecoxib-loaded PELA fibers in multi-layer membrane were uniform in size, randomly arrayed, very porous, and smooth without beads. Multi-layer membrane group had fewer peritendinous adhesions and better gliding than the PELA membrane group and control group in gross and histological observation. The similar mechanical characteristic and collagen expression of tendon repair site in the three groups indicated that the multi-layer membrane did not impair tendon healing. Taken together, our results demonstrated that such a biomimetic multi-layer sheath could be used as a potential strategy in clinics for promoting tendon gliding and preventing adhesion without poor tendon healing.     

Sustained Exposure of Substance P Causes Tendinopathy

Recently, neuromediators such as substance P (SP) have been found to be important factors in tendon homeostasis. Some studies have found SP to be the cause of inflammation and tendinopathy, whereas others have determined it to be a critical component of tendon healing. As demonstrated by these conflicting findings, the effects of SP on tendinopathy remain unclear. In this study, we hypothesized that the duration of SP exposure determines its effect on the tendons, with repetitive long-term exposure leading to the development of tendinopathy. First, we verified the changes in gene and protein expression using in vitro tenocytes with 10-day exposure to SP. SP and SP + Run groups were injected with SP in their Achilles tendon every other day for 14 days. Achilles tendons were then harvested for biomechanical testing and histological processing. Notably, tendinopathic changes with decreased tensile strength, as observed in the Positive Control, were observed in the Achilles in the SP group compared to the Negative Control. Subsequent histological analysis, including Alcian blue staining, also revealed alterations in the Achilles tendon, which were generally consistent with the findings of tendinopathy in SP and SP + Run groups. Immunohistochemical analysis revealed increased expression of SP in the SP group, similar to the Positive Control. In general, the SP + Run group showed worse tendinopathic changes. These results suggest that sustained exposure to SP may be involved in the development of tendinopathy. Future research on inhibiting SP is warranted to target SP in the treatment of tendinopathy and may be beneficial to patients with tendinopathy.     

The Role of MSCs and Cell Fusion in Tissue Regeneration

Regenerative medicine is concerned with the investigation of therapeutic agents that can be used to promote the process of regeneration after injury or in different diseases. Mesenchymal stem/stromal cells (MSCs) and their secretome—including extracellular vesicles (EVs) are of great interest, due to their role in tissue regeneration, immunomodulatory capacity and low immunogenicity. So far, clinical studies are not very conclusive as they show conflicting efficacies regarding the use of MSCs. An additional process possibly involved in regeneration might be cell fusion. This process occurs in both a physiological and a pathophysiological context and can be affected by immune response due to inflammation. In this review the role of MSCs and cell fusion in tissue regeneration is discussed.     

Ultrasonographic and Histological Correlation after Experimental Reconstruction of a Volumetric Muscle Loss Injury with Adipose Tissue

Different types of scaffolds are used to reconstruct muscle volume loss injuries. In this experimental study, we correlated ultrasound observations with histological findings in a muscle volume loss injury reconstructed with autologous adipose tissue. The outcome is compared with decellularized and porous matrix implants. Autologous adipose tissue, decellularized matrix, and a porous collagen matrix were implanted in volumetric muscle loss (VML) injuries generated on the anterior tibial muscles of Wistar rats. Sixty days after implantation, ultrasound findings were compared with histological and histomorphometric analysis. The muscles with an autologous adipose tissue implant exhibited an ultrasound pattern that was quite similar to that of the regenerative control muscles. From a histological point of view, the defects had been occupied by newly formed muscle tissue with certain structural abnormalities that would explain the differences between the ultrasound patterns of the normal control muscles and the regenerated ones. While the decellularized muscle matrix implant resulted in fibrosis and an inflammatory response, the porous collagen matrix implant was replaced by regenerative muscle fibers with neurogenic atrophy and fibrosis. In both cases, the ultrasound images reflected echogenic, echotextural, and vascular changes compatible with the histological findings of failed muscle regeneration. The ultrasound analysis confirmed the histological findings observed in the VML injuries reconstructed by autologous adipose tissue implantation. Ultrasound can be a useful tool for evaluating the structure of muscles reconstructed through tissue engineering.     

Cigarette Smoke Condensate Exposure Induces Receptor for Advanced Glycation End-Products (RAGE)-Dependent Sterile Inflammation in Amniotic Epithelial Cells

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.     

Bone Regeneration of a 3D-Printed Alloplastic and Particulate Xenogenic Graft with rhBMP-2

This study aimed to evaluate the bone regeneration capacity of a customized alloplastic material and xenograft with recombinant human bone morphogenetic protein-2 (rhBMP-2). We prepared hydroxyapatite (HA)/tricalcium phosphate (TCP) pure ceramic bone blocks made using a 3D printing system and added rhBMP-2 to both materials. In eight beagle dogs, a total of 32 defects were created on the lower jaws. The defective sites of the negative control group were left untreated (N group; 8 defects), and those in the positive control group were filled with particle-type Bio-Oss (P group; 12 defects). The defect sites in the experimental group were filled with 3D-printed synthetic bone blocks (3D group; 12 defects). Radiographic and histological evaluations were performed after healing periods of 6 and 12 weeks and showed no significant difference in new bone formation and total bone between the P and 3D groups. The 3D-printed custom HA/TCP graft with rhBMP-2 showed bone regeneration effects similar to that of particulate Bio-Oss with rhBMP-2. Through further study and development, the application of 3D-printed customized alloplastic grafts will be extended to various fields of bone regeneration.     

R-Spondin2, a Positive Canonical WNT Signaling Regulator,Controls the Expansion and Differentiation of Distal Lung Epithelial Stem/Progenitor Cells in Mice

The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/β-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.     

The Pathogenesis of Cardiac Fibrosis: A Review of Recent Progress

Fibrosis is defined as the excessive deposition of extracellular matrix (ECM) proteins in the interstitium. It is an essential pathological response to chronic inflammation. ECM protein deposition is initially protective and is critical for wound healing and tissue regeneration. However, pathological cardiac remodeling in excessive and continuous tissue damage with subsequent ECM deposition results in a distorted organ architecture and significantly impacts cardiac function. In this review, we summarized and discussed the histologic features of cardiac fibrosis with the signaling factors that control it. We evaluated the origin and characteristic markers of cardiac fibroblasts. We also discussed lymphatic vessels, which have become more important in recent years to improve cardiac fibrosis.     

Renal and Extra Renal Manifestations in Adult Zebrafish Model of Cystinosis

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.