Human adult, pediatric and microtia auricular cartilage harbor fibronectin-adhering progenitor cells with regenerative ear reconstruction potential.

Remaining challenges in auricular cartilage tissue engineering include acquiring sufficient amounts of regeneration-competent cells and subsequent production of high-quality neocartilage. Progenitor cells are a resident subpopulation of native cartilage, displaying a high proliferative and cartilage-forming capacity, yet their potential for regenerative medicine is vastly understudied. In this study, human auricular cartilage progenitor cells were newly identified in healthy cartilage and, importantly, in microtia-impaired chondral remnants. Their cartilage repair potential was assessed via in vitro 3D culture upon encapsulation in a gelatin-based hydrogel, and subsequent biochemical, mechanical, and histological analyses. Auricular cartilage progenitor cells demonstrate a potent ability to proliferate without losing their multipotent differentiation ability and to produce cartilage-like matrix in 3D culture. As these cells can be easily obtained through a non-deforming biopsy of the healthy ear or from the otherwise redundant microtia remnant, they can provide an important solution for long-existing challenges in auricular cartilage tissue engineering.

Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs.

Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material three-dimensional (3D) bioprinting strategies have the potential to resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge to date. In this study, we developed endothelial cell-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM), respectively. Human umbilical vein endothelial cell (HUVEC)-driven capillary networks started to form 2 d after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29%, and the number of microvessel junctions by 37% after 14 d, compared to bioinks with pro-angiogenic col-1 MFs. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a gellan gum microgel suspension bath. These 3D meniscus-like constructs were cultured up to 14 d, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications includingin vitromodels of vascular-to-avascular tissue interfaces, cancer progression, and for testing anti-angiogenic therapies.

Sensory Innervation of Human Bone: An Immunohistochemical Study to Further Understand Bone Pain.

Skeletal diseases and their surgical treatment induce severe pain. The innervation density of bone potentially explains the severe pain reported. Animal studies concluded that sensory myelinated A∂-fibers and unmyelinated C-fibers are mainly responsible for conducting bone pain, and that the innervation density of these nerve fibers was highest in periosteum. However, literature regarding sensory innervation of human bone is scarce. This observational study aimed to quantify sensory nerve fiber density in periosteum, cortical bone, and bone marrow of axial and appendicular human bones using immunohistochemistry and confocal microscopy. Multivariate Poisson regression analysis demonstrated that the total number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow, and cortical bone for all bones studied. Bone from thoracic vertebral bodies contained most sensory nerve fibers, followed by the upper extremity, lower extremity, and parietal neurocranium. The number of nerve fibers declined with age and did not differ between male and female specimens. Sensory nerve fibers were organized as a branched network throughout the periosteum. The current results provide an explanation for the severe pain accompanying skeletal disease, fracture, or surgery. Further, the results could provide more insight into mechanisms that generate and maintain skeletal pain and might aid in developing new treatment strategies. PERSPECTIVE: This article presents the innervation of human bone and assesses the effect of age, gender, bone compartment and type of bone on innervation density. The presented data provide an explanation for the severity of bone pain arising from skeletal diseases and their surgical treatment.

Instrumented cervical fusion in nine dogs with caudal cervical spondylomyelopathy.

OBJECTIVE: To report the long-term outcome of nine dogs treated for caudal cervical spondylomyelopathy (CCSM) with surgical spinal fusion. STUDY DESIGN: Short case series. ANIMALS: Nine large-breed dogs. METHODS: Medical records of dogs treated for disc-associated CCSM (2013-2016) were reviewed. The surgery objective was spinal distraction by implantation of a SynCage and fixation with two Unilock plates. Follow-up included the Helsinki pain score questionnaire, neurological grading, radiography, computed tomography (CT), and micro-CT (µCT) with subsequent histopathology (two dogs). RESULTS: Clinical follow-up was obtained between 9 and 51 months (27.4 ± 13.4 months). The Helsinki pain score and neurological Griffith score improved (P < .01) in all dogs and in eight of nine dogs, respectively. According to CT, the volume of bone (mean ± SD) through the cage was 79.5% ± 14.3%, including compact bone (53.0% ± 23.4%). Subsidence was seen in one of nine dogs. Implant failure was evident in four dogs, and plates were removed in two dogs. In seven of nine dogs, infraclinical pathology was observed in adjacent segment, associated with implants engaging adjacent intervertebral discs. Radiographic evidence of bony fusion between vertebral bodies was noted in all dogs. Spinal fusion was confirmed by µCT and histopathology in two cervical spine segments that became available at 22 and 40 months postoperatively. CONCLUSION: Instrumented spinal fusion in dogs with disc-associated CCSM resulted in owner satisfaction and radiographic evidence of interbody spinal fusion in all dogs. CLINICAL SIGNIFICANCE: The fusion distraction technique reported here can be used to achieve spinal fusion with a good long-term outcome.

Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients.

MSCs are known as multipotent mesenchymal stem cells that have been found capable of differentiating into various lineages including cartilage. However, recent studies suggest MSCs are pericytes that stimulate tissue repair through trophic signaling. Aimed at articular cartilage repair in a one-stage cell transplantation, this study provides first clinical evidence that MSCs stimulate autologous cartilage repair in the knee without engrafting in the host tissue. A phase I (first-in-man) clinical trial studied the one-stage application of allogeneic MSCs mixed with 10% or 20% recycled defect derived autologous chondrons for the treatment of cartilage defects in 35 patients. No treatment-related serious adverse events were found and statistically significant improvement in clinical outcome shown. Magnetic resonance imaging and second-look arthroscopies showed consistent newly formed cartilage tissue. A biopsy taken from the center of the repair tissue was found to have hyaline-like features with a high concentration of proteoglycans and type II collagen. DNA short tandem repeat analysis delivered unique proof that the regenerated tissue contained patient-DNA only. These findings support the hypothesis that allogeneic MSCs stimulate a regenerative host response. This first-in-man trial supports a paradigm shift in which MSCs are applied as augmentations or "signaling cells" rather than differentiating stem cells and opens doors for other applications. Stem Cells 2017;35:1984-1993.

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.

Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264.

Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis.

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.

Crosslinkable hydrogels derived from cartilage, meniscus, and tendon tissue.

Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.

Chondrogenic potential of articular chondrocytes depends on their original location.

OBJECTIVE: This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies. METHODS: Articular cartilage was obtained from Outerbridge grade III and IV cartilage lesions and from macroscopically healthy weight-bearing and nonweight-bearing (NWB) locations in the knee. Chondrocytes isolated from all locations were either pelleted directly (P0 pellets) or after expansion (P2 pellets) and analyzed for glycosaminoglycan (GAG), DNA, and cartilage-specific gene expression. Harvested cartilage samples and cultured pellets were also analyzed by Safranin O histology and immunohistochemistry for collagen I, II, and X. Immunohistochemical stainings were quantified using a computerized pixel-intensity staining segmentation method. RESULTS: After 4 weeks of culture, the P0 pellets derived from grade III or healthy weight-bearing chondrocytes contained more (p<0.015) GAG and GAG normalized per DNA compared to those from grade IV and NWB locations. After expansion, these differences were lost. Cartilage-specific gene expression was higher (p<0.04) in P0 pellets from grade III chondrocytes compared to grade IV chondrocytes. Semiquantitative immunohistochemistry showed a more intense (p<0.033) collagen I and X staining for grade IV debrided cartilage compared to grade III and weight-bearing cartilage. Also, collagen type X staining intensity was higher (p<0.033) in NWB cartilage compared to grade III and weight-bearing regions. CONCLUSION: Chondrocytes derived from debrided cartilage perform better than cells from the NWB biopsy site, however, this difference is lost upon expansion. Based thereon, the debrided defect cartilage could be a viable donor site for regenerative cartilage surgery.

Single-stage cell-based cartilage regeneration using a combination of chondrons and mesenchymal stromal cells: comparison with microfracture.

BACKGROUND: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix. PURPOSE: To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models. STUDY DESIGN: Controlled laboratory study. METHODS: Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs. RESULTS: The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment. CONCLUSION: The combination of chondrons and MSCs increased cartilage matrix formation, and this combination of cells was safely applied in a goat model for focal cartilage lesions, outperforming microfracture. CLINICAL RELEVANCE: This study describes the bench-to-preclinical development of a new cell-based regenerative treatment for focal articular cartilage defects that outperforms microfracture in goats. In addition, it is a single-step procedure, thereby making the expensive cell expansion and reimplantation of dedifferentiated cells, as in ACI, redundant.

Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well as for endochondral bone tissue engineering, as these approaches deal with, respectively, the inhibition or enhancement of hypertrophic chondrogenesis.

Hypoxia impedes vasculogenesis of in vitro engineered bone.

To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.

Replacement of the medial tibial plateau by a metallic implant in a goat model.

The purposes of the present study were to explore the surgical possibilities for replacement of the medial tibial plateau by a metallic implant in a large animal model and to examine the implications for the opposing cartilage. In six goats, the medial tibial plateau of the right knee was replaced by a cobalt-chromium implant, using polymethylmethacrylate bone cement for fixation. The unoperated left knee served as a control. At 26 weeks after surgery, the animals were killed, and the joints evaluated macroscopically. Cartilage quality was analyzed macroscopically and histologically. Glycosaminoglycan content, synthesis, and release were measured in tissue and medium. All animals were able to move and load the knees without any limitations. Macroscopic articular evaluation scores showed worsening 26 weeks after inserting the implant (p < 0.05). Macroscopic and histologic scores showed more cartilage degeneration of the opposing medial femoral condyle in the experimental knee compared to the control knee (p < 0.05). Higher glycosaminoglycan synthesis was measured at the medial femoral condyle cartilage in the experimental knees (p < 0.05). This study shows that the medial tibial plateau can be successfully replaced by a cobalt-chromium implant in a large animal model. However, considerable femoral cartilage degeneration of the medial femoral condyle was induced, suggesting that care must be taken introducing hemiarthroplasty devices in a human clinical setting for the treatment of postmeniscectomy cartilage degeneration of the medial tibial plateau.

Cartilage damage caused by metal implants applied for the treatment of established localized cartilage defects in a rabbit model.

The purpose of the current study was to investigate the feasibility of the application of defect-size femoral implants in a rabbit model of established cartilage defects and compare this treatment to microfracturing. In 31 New Zealand White rabbits, a medial femoral condyle defect was created in each knee. After 4 weeks, 3 animals were killed for defect baseline values. In the other 28 rabbits, knees were sham-operated, treated with microfracturing, or treated by placing an oxidized zirconium (OxZr) or cobalt-chromium (CoCr) implant (theta articulating surface 3.5 mm; fixating pin of 9.1 mm length). These animals were sacrificed 4 weeks after treatment. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry, and cartilage repair was scored microscopically. Cartilage quality was analyzed macroscopically and microscopically. Bone-implant contact was 63.2% +/- 3.2% for CoCr and 62.5% +/- 3.2% for OxZr. Cartilage defects did not show complete healing, nor during subsequent sham-surgery or microfracturing. For all treatments, considerable cartilage damage in the articulating medial tibia, and degeneration of lateral tibial and femoral cartilage was observed (p < 0.05). Both CoCr and OxZr implant-treated defects showed an increase of cartilage degeneration compared to microfracturing and sham-operated defects (p < 0.05). Although only a single short-term follow-up period was investigated in this study, caution is warranted using small metal implants as a treatment for established localized cartilage defects because, even after 4 weeks in this model, the metal implants caused considerable degeneration of the articulating surface.

Coronary end-to-side sleeve anastomosis using adhesive in off-pump bypass grafting in the pig.

BACKGROUND: In the exploration of facilitated coronary anastomosis strategies, we assessed a new octylcyanoacrylate adhesive in combination with a modified end-to-side sleeve anastomosis in off-pump bypass grafting in the pig. METHODS: Sleeve-adhesive anastomoses (n = 20) were evaluated intraoperatively, at 3 days (n = 4), and at 5 weeks (n = 16) in an off-pump, low (< or = 15 mL/min; n = 10) and high flow (approximately 60 mL/min; n = 10) porcine bypass model. All anastomoses were examined by flow measurement, angiography, and histology. RESULTS: Anastomosis construction took 8.5 minutes (6.7 to 10.2 minutes; median [15th to 85th percentile]). At 5 weeks, all anastomoses were fully patent (FitzGibbon grade A). The adhesive did not cause impaired vessel wall healing, but was surrounded by a focal acute and limited chronic (foreign body giant cells occasionally seen) inflammatory reaction at the adventitial application site. CONCLUSIONS: Octyl-cyanoacrylate tissue adhesive combined with end-to-side internal mammary to coronary artery sleeve anastomosis construction proved to be feasible, even in low bypass graft flow conditions (< or = 15 mL/min; prothrombotic milieu) in the pig and deserves interest in exploration of facilitated anastomosis strategies in coronary artery bypass grafting.