Fibrocartilage Tissue Research Articles

Implantation of autogenous meniscal fragments wrapped with a fascia sheath induces fibrocartilage regeneration in a large meniscal defect in sheep: A histological and biomechanical study

IntroductionCurrently, various studies have been reported to regenerate the meniscus tissue in a large defect after partial meniscectomy using biological or synthetic scaffolds with or without fibrochondrocytes. However, the clinical utility of those treatments has not been established as of yet. HypothesisPurposes of this study were to develop a sheep model to evaluate feasibility of this new surgical strategy to treat the irreparable meniscus injury, and to test the hypothesis that implantation of autogenous meniscal fragments wrapped with a fascia sheath may significantly induce fibrocartilage regeneration in a large meniscal defect in the sheep model. Methods and methodsTwenty Suffolk sheep were used. In each animal, an anterior 10-mm width of the right medial meniscus was resected. Then, the animals were divided into the following 2 groups. In Group I, the defect was enveloped with a fascia from the left thigh. In Group II, the resected meniscus fragmented into small pieces was grafted into the defect. Then the defect was enveloped with a fascia. In each group, 5 of 10 sheep were used for histological and biomechanical evaluations, respectively, at 12 weeks after surgery. ResultsIn Group I, the defect was incompletely filled with thin fibrous tissues, while fibrocartilage tissues rarely regenerated in the tissue. In Group II, all defects were completely filled with thick fibrocartilage tissues, which were richly stained with the safranin O staining. Both the gross and histological observation score of Group II was significantly (p=0.0005, p=0.0005) greater than that of Group I. Concerning the cross-sectional area of the regenerated tissue, Group II was significantly (p=0.0002) greater than Group I. In the biomechanical evaluation, the maximal load and the linear stiffness of the meniscus-tibia complex were significantly (p=0.0015, p=0.0283) greater in Group II than in Group I. DiscussionImplantation of autogenous meniscal fragments wrapped with a fascia sheath significantly induces fibrocartilage regeneration into a large meniscal defect in the sheep model. Level of evidenceNot applicable; Controlled Laboratory Study, Experimental in vivo study.

Inducing substances for chondrogenic differentiation of dental pulp stem cells in the conditioned medium of a novel chordoma cell line.

We successfully established a chordoma cell line, designated TSK-CHO1, derived from the clival chordoma. Currently, there is only one skull base chordoma cell line, UM-chor1, freely available to researchers. The established TSK-CHO1 cells were neoplastic, exhibited pleomorphic features, and secreted brachyury, as revealed by immunocytochemical staining or ELISA of conditioned medium (CM). Cells also secreted SOX9, which enhanced brachyury production. The CM of TSK-CHO1 cells promoted the production of hyaluronic acid and type II collagen during differentiation of human dental pulp stem cells (DPSCs) into fibrocartilage cells. Culture of DPSC pellets in a growth medium supplemented with 10% CM of TSK-CHO1 cells for 2weeks resulted in the induction of fibrocartilage tissue under normoxic conditions. Brachyury produced by TSK-CHO1 cells promoted the production of collagen type II, peculiar to cartilage, in a dose-dependent manner. The newly established skull base chordoma cell line, TSK-CHO1, is expected to be used for elucidating the pathogenesis of skull base chordoma and for investigating the mechanism underlying the production of fibrocartilage.

Equine Podotrochlear Apparatus - Histologic Characterization

Background: The navicular syndrome may be associated with alterations in other podotrochlear apparatus components, such as the deep digital flexor tendon, collareral sesamoid and distal sesamoid ligaments, podotrochlear bursa and distal sesamoid bone. However, the clinical significance and nature of these changes are not well understood, many of descriptive reports about distal sesamoid bone lesions are rarely accompanied by a complete and comprehensive comparison with animals of the control group. The aim of this study was to described histologically findings of the podrotrochlear apparatus components, allowing the understanding of the inserts and their microscopic appearance, thus providing the future recognize of their alterations.Materials, Methods & Results: Fourteen samples of the podotrochlear apparatus were taken out of 44 equine thoracic limbs specimens, separated at the radiocarpal joint of Crioulo and Thoroughbred horses, with an average age of 6.0-year-old, coming from a private clinic in southern Brazil. The thoracic limbs specimens were refrigerated at 4ºC at the clinic and then they were sent to the University Federal of Santa Maria (UFSM). Once at the University laboratory, the specimens were dissected to isolate the podotrochlear apparatus from each one. Subsequently, transversal and longitudinal samples from the distal sesamoid bone, deep digital flexor tendo, distal sesamoid ligament, colateral sesamoid ligament, were collected and podotrochlear bursa which were processed at the Veterinary Pathology Laboratory of the UFSM and University Federal of Santa Catarina (UFSC). The tissues samples were fixed in a 10% formaldehyde solution for 14 days and routinely processed for histology. The samples were sectioned at 3 µm and stained using the hematoxylin-eosin (H-E) routine method. The bone samples, after fixation, underwent a decalcified process in a formic acid-sodium citrate aqueous solution and routinely processed for histopathology. Histologic tendons evaluation showed that it is arranged in honeycombs fascicles in a transverse section observation. The tendinous fibers have a more compact and aligned collagen fibers arrangement due to the dense connective tissue. The synovial membrane of the podotrochlear bursa is composed of a cubic pseudo conjunctival epithelium arranged with a layer of synoviocytes forming villi towards the lumen. This characterization is very similar to that of the synovial joint membrane. The collateral sesamoid ligament fibers are not perfectly aligned, occurring at their confluence in some areas accompanied by the dissection of adipose tissue. The distal sesamoide ligament fibers, composed by of loose connective tissue associated with synoviocytes and vascular stroma. The articular surface of the distal sesamoid bone is arranged in 3 layers of chondrocytes embedded in a hyaline matrix. There was a clear differentiation between the subchondral bone area and the fibrocartilage tissue in the palmar aspect of the distal sesamoid bone.Discussion: The morphophysiological characterization of the podotrochlear structures inside the hoof capsule it is important for the future recognition of abnormalities and the possible hypothesis that originates the podotrochlear syndrome. These changes have great clinical relevance and very often associated of distal limb lameness in horses. The normal macroscopic, ultrasonographic and radiographic images along with the histomorphometric study of the podotrochlear structures have contributed for the morphophysiological comprehension and consequently future interpretation of the podotrochlear structures diseases.Keywords: podotrochlear apparatus, distal sesamoide bone, deep digital flexor tendon, navicular syndrome, histological findings.

Plasma-assisted multiscale topographic scaffolds for soft and hard tissue regeneration

The design of transplantable scaffolds for tissue regeneration requires gaining precise control of topographical properties. Here, we propose a methodology to fabricate hierarchical multiscale scaffolds with controlled hydrophilic and hydrophobic properties by employing capillary force lithography in combination with plasma modification. Using our method, we fabricated biodegradable biomaterial (i.e., polycaprolactone (PCL))-based nitrogen gas (N-FN) and oxygen gas plasma-assisted flexible multiscale nanotopographic (O-FMN) patches with natural extracellular matrix-like hierarchical structures along with flexible and controlled hydrophilic properties. In response to multiscale nanotopographic and chemically modified surface cues, the proliferation and osteogenic mineralization of cells were significantly promoted. Furthermore, the O-FMN patch enhanced regeneration of the mineralized fibrocartilage tissue of the tendon–bone interface and the calvarial bone tissue in vivo in rat models. Overall, the PCL-based O-FMN patches could accelerate soft- and hard-tissue regeneration. Thus, our proposed methodology was confirmed as an efficient approach for the design and manipulation of scaffolds having a multiscale topography with controlled hydrophilic property.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

Three-dimensional ultrasound for knee osteophyte depiction: a comparative study to computed tomography

PurposeOsteophytes are common radiographic markers of osteoarthritis. However, they are not accurately depicted using conventional imaging, thus hampering surgical interventions that rely on pre-operative images. Studies have shown that ultrasound (US) is promising at detecting osteophytes and monitoring the progression of osteoarthritis. Furthermore, three-dimensional (3D) ultrasound reconstructions may offer a means to quantify osteophytes. The purpose of this study was to compare the accuracy of osteophyte depiction in the knee joint between 3D US and conventional computed tomography (CT).MethodsEleven human cadaveric knees were pre-screened for the presence of osteophytes. Three osteoarthritic knees were selected, and then, 3D US and CT images were obtained, segmented, and digitally reconstructed in 3D. After dissection, high-resolution structured light scanner (SLS) images of the joint surfaces were obtained. Surface matching and root mean square (RMS) error analyses of surface distances were performed to assess the accuracy of each modality in capturing osteophytes. The RMS errors were compared between 3D US, CT and SLS models.ResultsAverage RMS error comparisons for 3D US versus SLS and CT versus SLS models were 0.87 mm ± 0.33 mm (average ± standard deviation) and 0.95 mm ± 0.32 mm, respectively. No statistical difference was found between 3D US and CT. Comparative observations of imaging modalities suggested that 3D US better depicted osteophytes with cartilage and fibrocartilage tissue characteristics compared to CT.ConclusionUsing 3D US can improve the depiction of osteophytes with a cartilaginous portion compared to CT. It can also provide useful information about the presence and extent of osteophytes. Whilst algorithm improvements for automatic segmentation and registration of US are needed to provide a more robust investigation of osteophyte depiction accuracy, this investigation puts forward the potential application for 3D US in routine diagnostic evaluations and pre-operative planning of osteoarthritis.

Changes in the avascular area of the meniscus using mesenchymal stem cells and growth plate chondrocytes in a pig model.

Menisci are wedge-shaped cartilage discs that are divided into two parts: the avascular and vascular regions. They are formed by fibrocartilage tissue, which contains round cartilage-like cells and extracellular matrix. Meniscus injury in animals is a common orthopedic problem, but data on the natural healing process mainly deals with the vascular zone. The healing processes in the avascular zone of the meniscus are significantly limited. Thus, this study aimed to evaluate autologous growth plate chondrocytes' impact on the healing process of a damaged meniscus in the avascular zone based on a growing animal model. The study group consisted of 10 pigs at about three months of age. From each animal, chondrocytes from the iliac growth plate and from concentrated bone marrow were taken. Knee joints were divided into right (R) and left (L). The medial meniscus of the R knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates (nCHON). The medial meniscus of the L knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates supplemented with immature chondrocytes isolated from growth plates (wCHON). The meniscus was damaged in the avascular zone in both knee joints. Followingly, the damaged part of the meniscus was filled with a scaffold with cells from the concentrated bone marrow and from growth plate chondrocytes. In the control group, a scaffold with concentrated bone marrow cells was used. After three months the animals were euthanized and preparations (microscopic slides) were made from the meniscus' damaged part. A qualitative and quantitative analysis have been prepared. The wCHON group in comparison with the nCHON group showed a statistically significantly higher number of fusiform cells on the surface of the graft as well as better healing of the graft. In addition, the degree of vascularization was higher in specimens from the wCHON group than in the nCHON group. The results of our research on immature pig knees revealed that mesenchymal stem cell and growth plate chondrocytes could be treated as the cell source for meniscus reconstruction, and growth plate chondrocytes enhance healing processes in the avascular zone of the injured meniscus.

Transcriptional profiling of mESC-derived tendon and fibrocartilage cell fate switch

The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFβ and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFβ-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.

Lumbar Interbody Fusion Conducted on a Porcine Model with a Bioresorbable Ceramic/Biopolymer Hybrid Implant Enriched with Hyperstable Fibroblast Growth Factor 2.

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p < 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

Quantitative MRI T2 Mapping is Able to Assess Tissue Quality After Reparative and Regenerative Treatments of Osteochondral Lesions of the Talus.

BackgroundQuantitative MRI has potential for tissue characterization after reparative and regenerative surgical treatment of osteochondral lesions of the talus (OCLTs). However available data is inconclusive and quantitative sequences can be difficult to implement in real‐time clinical application.PurposeTo assess the potential of T2 mapping in discriminating articular tissue characteristics after reparative and regenerative surgery of OCLTs in real‐world clinical settings.Study TypeObservational and prospective cohort study.Population15 OCLT patients who had received either reparative treatment with arthroscopic microfracture surgery (MFS) for a grade I lesion or regenerative treatment with bone marrow derived cell transplantation (BMDCT) for a grade II lesion.Field Strength/Sequence1.5 T, proton density weighted TSE, T2‐weighted true fast imaging with steady‐state‐free precession and multi‐echo T2 mapping sequences.AssessmentPatients were evaluated at a minimum postoperative follow‐up of 24 months. T2 maps of the ankle were generated and the distribution of T2 values was analyzed in manually identified volumes of interest (VOIs) for both treated lesions (TX) and healthy cartilage (CTRL). The amount of fibrocartilage, hyaline‐like and remodeling tissue in TX VOIs was obtained, based on T2 thresholds from CTRL VOIs.Statistical TestsFisher's exact test for categorical data, nonparametric Mann–Whitney U test for continuous data. The statistical significance level was P < 0.05.ResultsFrom CTRL VOI analysis, T2 < 25 msec, 25 msec ≤ T2 ≤ 45 msec, and T2 > 45 msec were considered as representative for fibrocartilage, hyaline‐like and remodeling tissue, respectively. Tissue composition of the two treatment groups was different, with significantly more fibrocartilage (+28%) and less hyaline‐like tissue (−15%) in MFS than in BMDCT treated lesions. No difference in healthy tissue composition was found between the two groups (P = 0.75).Data ConclusionsT2 mapping of surgically treated OCLTs can provide quantitative information about the type and amount of newly formed tissue at the lesion site, thereby facilitating surgical follow‐up in a real‐word clinical setting.Level of Evidence2Technical EfficacyStage 3

Shear viscoelasticity of electrospinning PCL nanofibers reinforced alginate hydrogels

Articular cartilage has limited self-repair capacity due to the lack of vascularization, innervation and lymphatic networks. Biomimetic scaffolds with features of the extracellular matrix (ECM) of cartilage are advantageous to repair the injured cartilage tissue, but it remains a challenge to regulate its shear viscoelasticity to meet the needs of applications as articular cartilages. Fiber reinforced hydrogel is of great significance for their clinical application as cartilage tissue engineering scaffolds, especially for repairing the fibrocartilage tissue like meniscus or temporomandibular joint disc. In order to promote the shear viscoelasticity of alginate hydrogels, which was seldom studied, electrospinning PCL nanofiber layers were added into the alginate hydrogels to prepare PCL nanofibers reinforced alginate hydrogel composites (PNRAHCs). Compared with neat alginate hydrogel scaffolds, the PNRAHCs presented coral-like structure and spider web-like structure, and some PCL nanofibers form reinforced fiber bundles. Those special structures make the PNRAHCs have higher porosity, higher shear storage modulus and higher shear loss modulus than the neat alginate hydrogels, indicating better shear mechanical properties. They have the potential to be applied as the scaffolds to repair fibrocartilage tissues.

Distinct ex vivo biomarker profiles of calcified and non-calcified acetabular labrum tissues in primary hip OA

Purpose: Hip OA has a prevalence of 7.4% in individuals older than 60 years and involves structural degeneration of several joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. The latter is a ring-form fibrocartilage tissue that plays a fundamental role in hip joint stability and articular cartilage homeostasis. Degeneration of the labrum involves chondrocyte apoptosis, macrophage infiltration and calcification and is thought to promote progression of primary hip OA. Moreover, labrum is a vascularized and innervated tissue and therefore strongly implied in hip OA pain. While several histological studies have documented morpological tissue changes, biochemical changes have not been extensively reported. In this study, we characterized biomarker secretion of explanted human labrum tissues under basal and inflammatory conditions. Methods: Intact labrum specimens were obtained from patients undergoing total hip arthroplasty (n=11, mean age 65.5, 3 women) and dissected in equal-sized samples (200-400 mg wet weight). Labrum tissues were subjected to explant culture for one week in the presence or absence of an inflammatory stimulus (1 ug/mL LPS). To evaluate the role of TGF-beta signaling in labrum tissue metabolism, samples were treated with 10 uM SB-505124 (TGF-beta type I receptor inhibitor) in the presence or absence of LPS. Secretion of Aggrecan (ACAN), Cartilage Oligomeric Matrix Protein (COMP), MMP13, Pro-Collagen-Ia and IL-6 was assessed by ELISA. Tissue proteoglycans and calcification were evaluated by whole mount Alcian blue/Alizarin red staining followed by ethyl cinnamate-based optical clearing and fluorescence microscopy. Results: Whole mount staining for proteoglycans and calcium revealed different degrees of calcification of labrum tissues (Figure). Five samples contained numerous calcified nodules and one sample was completely ossified. Non-calcified specimens displayed strong proteoglycan tissue staining interspersed with fibrous tissue. Labrum tissues secreted low levels of IL-6 and ACAN (5-50 ng/gr tissue), moderate levels of MMP13 and COMP (100-300 ng/gr tissue) and high levels of Pro-Collagen-Ia (∼900 ng/gr tissue). Subgroup analysis revealed significantly higher Pro-Collagen-Ia (1170+230 vs. 596+305) and MMP13 (157+22 vs. 60+15) secretion by calcified labrum specimens. Inflammatory stimulation led to a ∼10-fold upregulation of IL-6 and ∼2-fold dowregulation of Pro-Collagen-Ia secretion, in both non-calcified and calcified specimens. Neither basal expression, nor effects of LPS stimulation were mediated through TGF-beta signaling. Conclusions: Biomarker profiling of degenerative acetabular labrum tissues revealed distinct secretion patterns in non-decalcified and calcified specimens, displaying elevated Pro-Collagen-Ia and MMP13 secretion by the latter. In contrast to articular cartilage, regulation of IL-6 and Pro-Collagen-Ia was not TGF-beta, but rather TLR4-dependent and calcification appeared IL-6-independent. Whether labrum fibrochondrytes or infiltrated macrophages are crucially involved in these effects remains to be elucidated. Due to its close proximity to articular cartilage, secreted mediators from degenerated labrum might contribute to or even accelerate the development of hip OA.

Fibrin Glue-Kartogenin Complex Promotes the Regeneration of the Tendon-Bone Interface in Rotator Cuff Injury.

Objective Rotator cuff injury healing is problematic because the tendon-bone junction often forms cicatricial tissues, rather than fibrocartilage, which leads to mechanical impairment and is prone to redamage. Kartogenin (KGN) is a newly discovered small molecule compound which can induce cartilage formation through chondrogenesis of endogenous mesenchymal stem cells. Methods In this study, we used KGN with fibrin glue (FG) to repair the rotator cuff injury by promoting the formation of fibrocartilage at the tendon to bone interface. Firstly, we assessed the release rate of KGN from the FG-KGN complex and then created a rabbit rotator cuff tendon graft-bone tunnel model. The rabbits received saline, FG-KGN, or FG injections onto the tendon to bone interface after injury. Shoulder tissues were harvested at 6 and 12 weeks, and the sections were stained with HE and Safranin O/Fast green. The samples were assessed by histologic evaluation and biomechanical testing. Synovial mesenchymal stem cells derived from the synovial tissue around the rotator cuff were harvested for western blotting and qRT-PCR analysis. Results KGN was released rapidly from the FG-KGN complex during first 4 hrs and followed by a slow release until 7 days. The tendon graft-bone interface in the control (saline) group and the FG group was filled with scar tissue, rather than cartilage-like tissue, and only a small number of chondrocytes were found at the adjacent bone surface. In the FG-KGN group, the tendon to bone interface was fully integrated and populated by chondrocytes with proteoglycan deposition, indicating the formation of fibrocartilage-like tissues. At 12 weeks, the maximum tensile strength of the FG-KGN group was significantly higher than that of the FG and control groups (P < 0.01). The RNA expression levels of tendinous genes such as Tenascin C and the chondrogenic gene Sox-9 were substantially elevated in SMSCs treated with the FG-KGN complex compared to the other two groups. Conclusion These results indicated that fibrin glue is an effective carrier for KGN, allowing for the sustained release of KGN. The FG-KGN complex could effectively promote the regeneration and formation of fibrocartilage tissue of the tendon-bone interface in the rabbit rotator cuff tendon graft-bone tunnel model.

Cell-free scaffolds functionalized with bionic cartilage acellular matrix microspheres to enhance the microfracture treatment of articular cartilage defects.

Microfracture surgery remains the most popular treatment for articular cartilage lesions in the clinic, but often leads to the formation of inferior fibrocartilage tissue and damage to subchondral bone. To overcome these problems, extracellular matrix (ECM) scaffolds derived from decellularized natural cartilaginous tissues were introduced and showed excellent biological properties to direct the differentiation of bone marrow stem cells. However, besides the limited allogenic/allogenic supply and the risk of disease transfer from xenogeneic tissues, the effectiveness of ECM scaffolds always varied with a high variability of natural tissue quality. In this study, we developed composite scaffolds functionalized with a cell-derived ECM source, namely, bionic cartilage acellular matrix microspheres (BCAMMs), that support the chondrogenic differentiation of bone marrow cells released from microfracture. The scaffolds with BCAMMs at different developmental stages were investigated in articular cartilage regeneration and subchondral bone repair. Compared to microfracture, the addition of cell-free BCAMM scaffolds has demonstrated a great improvement of regenerated cartilage tissue quality in a rabbit model as characterized by a semi-quantitative analysis of cells, histology and biochemical assays as well as micro-CT images. Moreover, the variation in ECM properties was found to significantly affect the cartilage regeneration, highlighting the challenges of homogenous scaffolds in working with microfracture. Together, our results demonstrate that the biofunctionalized BCAMM scaffold with cell-derived ECM shows great potential to combine with microfracture for clinical translation to repair cartilage defects.

Electrodeposition of calcium phosphate onto polyethylene terephthalate artificial ligament enhances graft-bone integration after anterior cruciate ligament reconstruction

It is a big challenge to develop a polyethylene terephthalate (PET) artificial ligament with excellent osteogenetic activity to enhance graft-bone integration for ligament reconstruction. Herein, we evaluated the effect of biomineralization (BM) and electrodeposition (ED) method for depositing calcium-phosphate (CaP) on the PET artificial ligament in vitro and in vivo. Scanning electron microscopy and energy-dispersive X-Ray spectrometer mapping analysis revealed that the ED-CaP had more uniform particles and element distribution (Ca, P and O), and thermogravimetric analysis showed there were more CaP on the PET/ED-CaP than the PET/BM-CaP scaffold. Moreover, the hydrophilicity of PET scaffolds was significantly improved after CaP deposition. In vitro study showed that CaP coating via BM or ED method could improve the attachment and proliferation of MC3T3-E1 cells, and ED-CaP coating significantly increased osteogenic differentiation of the cells, in which the Wnt/β-catenin signaling pathway might be involved. In addition, radiological, histological and immunohistochemical results of in vivo study in a rabbit anterior cruciate ligament (ACL) reconstruction model demonstrated that the PET/BM-CaP and PET/ED-CaP scaffolds significantly improved graft-bone integration process compared to the PET scaffold. More importantly, larger areas of new bone ingrowth and the formation of fibrocartilage tissue were observed at 12 weeks in the PET/ED-CaP group, and the biomechanical tests showed increased ultimate failure load and stiffness in PET/ED-CaP group compared to PET/BM-CaP and PET group. Therefore, ED of CaP is an effective strategy for the modification of PET artificial ligament and can enhance graft-bone integration both in vitro and in vivo.

The Inner Annulus Fibrosus Encroaches on the Nucleus Pulposus in the Injured Mouse Tail Intervertebral Disc.

The aim was to identify the source of cells within the center of the abnormal fibrocartilage tissue of the degenerative intervertebral disc after injury. Cross-breeding of mice with an inducible type II promoter collagen construct (Col2CreER) to Rosa26-TdTomato mice has been shown to result in Cre-recombinase activity and Tomato expression in inner annulus fibrosus cells after tamoxifen injection. To investigate the role of the inner annulus fibrosus in the intervertebral disc injury response, tail intervertebral discs of Col2CreER/tdTomato mice were punctured with a needle and examined 1-4 wks after injury. N-cadherin was examined by immunostaining. After the injury, the fibrocartilage in the degenerative intervertebral disc consisted of residual diseased nucleus pulposus cells and encroaching inner annulus fibrosus cells. The residual nucleus pulposus cells had lost their epithelial cell-like morphology and instead became oval shaped, with reduced adhesion to neighboring nucleus pulposus cells. This change in cellular morphology coincided with a loss of N-cadherin, which contributes to maintenance of healthy nucleus pulposus cell morphology. As expected, injured tail intervertebral discs showed reduced compressive properties as determined by biomechanical assessments. The cellular composition of the degenerative intervertebral disc has been defined here, which is an important step in developing future treatments.

Effects of lentivirus-mediated insulin-like growth factor 1 and platelet derived growth factor genes on nucleus pulposus tissue of human degenerated intervertebral disc

To observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP. Human degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot. HE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group ( P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group ( t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A ( P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant ( P<0.05). Both IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.

Using Histological and Chemical Methods for Detection of Unauthorized Tissues Addition in Emulsion Type Meat Product

Total twenty different processed meat plant producing emulsion type sausage were histologically and chemically examined for detection of adulteration with unauthorized tissues. Results revealed that samples were adulterated with different types of animal tissues included; hyaline cartilage, tendon, spongy bone, peripheral nerve trunk, basophilic matrix, lymphatic tissue, fascia, fibrocartilage and vascular tissue. Moreover, these samples were adulterated Also, adulterated with plant tissue included; plant stem, leaves and root. Chemical analysis showed a significant difference in their chemical composition (moisture, fat, protein, ash and calcium) content. Moisture and fat content varied around the permissible limit of E.S.S. while low protein, high ash and calcium content was detected in the examined samples. Therefore, Histological and chemical examinations can be used as reliable methods to detect adultration using unauthorized addition of both animal and plant tissues in processed meat product samples which revealed a high level of falsification.

Biomineralizaion of hydroxyapatite on polyethylene terephthalate artificial ligaments promotes graft-bone healing after anterior cruciate ligament reconstruction: An in vitro and in vivo study.

The purpose of the present study is to modify the polyethylene terephthalate ligament with hydroxyapatite via biomineralization and to investigate its effect on graft-bone healing. After biomineralization of hydroxyapatite, the surface characterization of polyethylene terephthalate ligament was examined by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and water contact angle measurements. The compatibility and osteoinduction, along with the underlying signaling pathway involved of hydroxyapatite-polyethylene terephthalate ligament, were evaluated in vitro. Moreover, a rabbit anterior cruciate ligament reconstruction model was established, and the polyethylene terephthalate or hydroxyapatite-polyethylene terephthalate artificial ligament was implanted into the knee. The micro-computed tomography analysis, histological, and immunohistochemical examination as well as biomechanical test were performed to investigate the effect of hydroxyapatite coating in vivo. The results of scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction showed that the hydroxyapatite was successfully deposited on the polyethylene terephthalate ligament. Water contact angle of the hydroxyapatite-polyethylene terephthalate group was significantly smaller than that of the polyethylene terephthalate group. The in vitro study showed that hydroxyapatite coating significantly improved adhesion and proliferation of MC3T3-E1 cells. The osteogenic differentiation of cells was also enhanced through the activation of ERK1/2 pathway. The micro-computed tomography, histological, and immunohistochemical results showed that biomineralization of hydroxyapatite significantly promoted new bone and fibrocartilage tissue formation at 12 weeks postoperatively. Moreover, the failure load and stiffness in the hydroxyapatite-polyethylene terephthalate group were higher than that in the polyethylene terephthalate group. Therefore, biomineralizaion of hydroxyapatite enhances the biocompatibility and osseointegration of the polyethylene terephthalate artificial ligament, thus promoting graft-bone healing for anterior cruciate ligament reconstruction through the activation of ERK1/2 pathway.

Augmentation of Tendon Graft–Bone Tunnel Interface Healing by Use of Bioactive Platelet-Rich Fibrin Scaffolds

Background: Platelet-rich fibrin (PRF) is a bioactive biomaterial wherein cytokines are enmeshed within the interconnecting fibrin network. PRF can be fabricated into a patch to augment healing of the interface between a tendon graft and bone tunnel. Hypothesis: The bioactivity of a PRF scaffold is preserved after PRF is mechanically compressed into a patch. A bioactive PRF patch could promote the incorporation of a tendon graft within the bone tunnel through the formation of a tendon-bone healing zone composed of both fibrocartilaginous tissue and new bone. Study Design: Controlled laboratory study. Methods: Bioactivity of PRF was evaluated through treatment of rabbit tenocytes with PRF-conditioned medium and cultivation of cells on a PRF patch. Cellular morphologic features, viability, and differentiation were analyzed accordingly. In an animal study, a rabbit tendon-bone healing model was established through use of New Zealand White rabbits. The implanted tendon graft was enveloped circumferentially with a bioactive PRF patch before being pulled through a bone tunnel in the proximal tibia. Micro–computed tomography (micro-CT) imaging and histological and biomechanical analyses of the tendon-bone interface were performed at 12 weeks postoperatively. Results: PRF improved the viability of the cultured tenocytes. The effects of PRF on in vitro mineralization of tenocytes were comparable with the effects of standard culture medium. The gene expressions of type I collagen and osteopontin were upregulated upon PRF treatment. For the in vivo study, micro-CT images revealed significant new bone synthesis at the tendon-bone interface in the PRF-enveloped group. The tendon-bone healing zone was characterized by abundant fibrocartilage tissue and new bone formation as demonstrated by histological analysis. Biomechanical testing showed significantly higher ultimate loads in the PRF-enveloped group. Conclusion: Bioactive PRF could effectively augment healing of tendon graft to bone by inducing the formation of a transitional tendon-bone healing zone composed of fibrocartilage and bone. Clinical Relevance: Complete healing of the tendon graft in the bone tunnel is a prerequisite for successful ligament reconstruction, which would allow early and aggressive rehabilitation and rapid return to preinjury activity level. From a translational standpoint, the PRF-augmented healing in this rabbit animal model showed a promising biological approach to enhance tendon graft to bone healing via promotion of the functional anchorage between the 2 different materials.