Mattie H.P. van Rijen

Identification of Cell Surface-Specific Markers to Target Human Nucleus Pulposus Cells Expression of Carbonic Anhydrase XII Varies With Age and Degeneration

OBJECTIVE Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. METHODS We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. RESULTS Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. CONCLUSION CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

Zonal Chondrocyte Subpopulations Reacquire Zone-Specific Characteristics During In Vitro Redifferentiation

Background If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Hypothesis Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Study Design Controlled laboratory study. Methods Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes). Results Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone. Conclusion Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion. Clinical Relevance Appreciation of the zonal differences can lead to important advances in cartilage tissue engineering. Findings support the use of hydrogels such as alginate for engineering zonal cartilage constructs.

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

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.

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 (∅︁ 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.

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

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.

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.

The groove model of tibia-femoral osteoarthritis in the rat.

Several experimental models of osteoarthritis in rats are used to study the pathophysiology of osteoarthritis. Many mechanically induced models have the limitation that permanent joint instability is induced by, for example, ligament transection or meniscal damage. This permanent instability will counteract the potential beneficial effects of therapy. The groove model of osteoarthritis uses a one‐time trigger, surgically induced cartilage damage on the femoral condyles, and has been validated for the canine tibia‐femoral compartment. The present study evaluates this model for the rat knee joint. The articular cartilage of the weight bearing surface of both femoral condyles and trochlea were damaged (grooved) without damaging the underlying subchondral bone. Severity of joint degeneration was histologically assessed, in addition to patella cartilage damage, and subchondral bone characteristics by means of (contrast‐enhanced) micro‐CT. Mild histological degeneration of the surgically untouched tibial plateau cartilage was observed in addition to damage of the femoral condyles, without clear synovial tissue inflammation. Contrast enhanced micro‐CT demonstrated proteoglycan loss of the surgically untouched patella cartilage. Besides, a more sclerotic structure of the subchondral bone was observed. The tibia‐femoral groove model in a rat results in mild knee joint degeneration, without permanent joint instability and joint inflammation. This makes the rat groove model a useful model to study the onset and progression of post‐traumatic non‐inflammatory osteoarthritis, creating a relatively sensitive model to study disease modifying osteoarthritic drugs.

Prolonged inhibition of inflammation in osteoarthritis by triamcinolone acetonide released from a polyester amide microsphere platform

&NA; Controlled biomaterial‐based corticosteroid release might circumvent multiple injections and the accompanying risks, such as hormone imbalance and muscle weakness, in osteoarthritic (OA) patients. For this purpose, microspheres were prepared from an amino acid‐based polyester amide (PEA) platform and loaded with triamcinolone acetonide (TAA). TAA loaded microspheres were shown to release TAA for over 60 days in PBS. Furthermore, the bioactivity lasted at least 28 days, demonstrated by a 80–95% inhibition of PGE2 production using TNF&agr;‐stimulated chondrocyte culture, indicating inhibition of inflammation. Microspheres loaded with the near infrared marker NIR780‐iodide injected in healthy rat joints or joints with mild collagenase‐induced OA showed retention of the microspheres up till 70 days after injection. After intra‐articular injection of TAA‐loaded microspheres, TAA was detectable in the serum until day seven. Synovial inflammation was significantly lower in OA joints injected with TAA‐loaded microspheres based on histological Krenn scores. Injection of TAA‐loaded nor empty microspheres had no effect on cartilage integrity as determined by Mankin scoring. In conclusion, the PEA platform shows safety and efficacy upon intra‐articular injection, and its extended degradation and release profiles compared to the currently used PLGA platforms may render it a good alternative. Even though further in vivo studies may need to address dosing and readout parameters such as pain, no effect on cartilage pathology was found and inflammation was effectively lowered in OA joints. Graphical abstract Figure. No caption available.