Prophylaxis of implant-related infections by local release of vancomycin from a hydrogel in rabbits.

Local prophylaxis with antibiotic-loaded bone cement is a successful method to prevent post-operative infections in patients receiving orthopaedic implants. No comparable method is available for uncemented implants. Therefore, a hydrogel consisting of hyaluronic and polylactic acids was evaluated in a rabbit model for delivery of antimicrobial agents to prevent post-operative infections. In a pilot study, the suitability of the in vivo model was assessed by testing the hydrogel as carrier material for antimicrobial agents.In the main study, the antimicrobial-agent-loaded hydrogel was evaluated for infection prophylaxis. Rabbits received a titanium rod intramedullary in the tibia after contamination with Staphylococcus aureus. The rods were coated with unloaded hydrogel (Gel), hydrogel loaded with 2 % (Van2) or 5 % vancomycin (Van5), bioactive glass (BAG) or N-acetyl-L-cysteine (NAC). To analyse the infection severity after 28 d, histopathological, bacteriological, micro-computed tomographic and haematological analyses were performed. In the pilot study, the Van5 group had less infection (0/6 infected) as compared to the Gel group (5/5, p = 0.000) and the in vivo model was deemed suitable. In the main study, in the Van2 and Van5 groups, the number of infected animals was lower [1/6 (p = 0.006) and 2/6 (p = 0.044) infected, respectively]. In contrast, BAG and NAC groups showed no infection reduction (5/6 both groups, p = 0.997). The hydrogel can be used as a local carrier of vancomycin for prophylaxis of implant-related infections.The present study showed promising results for local delivery of antibacterial agents by hydrogel to prevent implant-related infections.

The role of bacterial stimuli in inflammation-driven bone formation.

Immune cells and their soluble factors regulate skeletal cells during normal bone regeneration and pathological bone formation. Bacterial infections can trigger immune responses that activate pro-osteogenic pathways, but these are usually overshadowed by osteolysis and concerns of systemic inflammation. The aim of this study was to determine whether the transient local inflammatory reaction to non-viable bacterial immune agonists could lead to favourable new bone formation. In a series of rabbit studies, as proof-of-concept, how tibial intramedullary injection of viable or killed bacterial species affected bone remodelling and new bone formation was determined. Application of killed bacteria led to considerable new bone formation after 4 weeks, without the prolonged systemic inflammation and exaggerated bone lysis seen with active infection. The osteo-immunomodulatory effects of various species of killed bacteria and the dose response relationship were subsequently screened in ectopically-implanted ceramic scaffolds. Histomorphometry after 8 weeks showed that a relatively low dose of killed bacteria enhanced ectopic bone induction. Moreover, lipoteichoic acid - the bacterial cell-wall derived toll-like-receptor (TLR)-2 activator - was identified as an osteo-stimulatory factor. Collectively, the data indicated that bacterial stimuli could be harnessed to stimulate osteogenesis, which occurs through a synergy with osteoinductive signals. This finding holds promise for the use of non-viable bacteria, bacterial antigens, or their simplified analogues as immuno-modulatory bone regenerating tools in bone biomaterials.

Ethics in musculoskeletal regenerative medicine; guidance in choosing the appropriate comparator in clinical trials.

BACKGROUND: Regenerative Medicine (RM) techniques aimed at the musculoskeletal system are increasingly translated to clinical trials and patient care. This revolutionary era in science raises novel ethical challenges. One of these challenges concerns the appropriate choice of the comparator in (randomized controlled) trials, including the ethically contentious use of sham procedures. To date, only general guidelines regarding the choice of the comparator exist. OBJECTIVE: To provide specific guidelines for clinical trial comparator choice in musculoskeletal RM. METHODS: In this manuscript, we discuss the ethics of comparator selection in RM trials. First, we make a classification of RM interventions according to different health states from disease prevention, return to normal health, postponing RM treatment, supplementing RM treatment, substituting RM treatment, improving RM outcome, and slowing progression. Subsequently, per objective, the accompanying ethical points to consider are evaluated with support from the available literature. RESULTS: a sham procedure is demonstrated to be an ethically acceptable comparator in RM trials with certain objectives, but less appropriate for musculoskeletal RM interventions that aim at preventing disease or substituting a surgical treatment. The latter may be compared to 'standard of care'. CONCLUSION: From a scientific perspective, choosing the correct comparator based on ethical guidelines is a step forward in the success of musculoskeletal RM.

Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation.

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day 10. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the bone volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with bone-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in bone formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved bone graft substitutes.

Bone formation by heterodimers through non-viral gene delivery of BMP-2/6 and BMP-2/7.

Non-viral gene delivery is a safe technique to release sustained physiologic dosages of bone morphogenetic protein (BMP). Co-delivery of multiple BMPs can result in the formation of more potent BMP heterodimers. In this study, non-viral co-delivery of BMP-2/6 and BMP-2/7, as a mean to produce heterodimers, was assessed. Goat MSCs were non-virally transfected with plasmid DNA encoding BMP isoforms (pBMP) known to be relevant for osteogenesis: BMP-2, -6 or -7. As a result, BMP-2, -6 and -7 were produced and detectable for up to 14 d and their combined delivery (pBMP-2 with pBMP-6 or pBMP-7) was used to create BMP-2/6 and BM-2/7 heterodimers. Formation and secretion of the heterodimer proteins was validated by sandwich enzyme-linked immunosorbent assay (ELISA). Produced BMPs and heterodimers were biologically active, as confirmed by differentiation of reporter cells and MSCs. To assess bone formation, transfected MSCs were seeded on to ceramic scaffolds and implanted subcutaneously into nude mice. Bone formation was significantly enhanced in the pBMP-2/6 condition and a trend for more bone formation was observed in the pBMP-2/7 and pBMP-6 homodimer condition. No bone was found in the pBMP-2, pBMP-7 or control condition. In conclusion, simultaneous delivery of pBMP-2 with pBMP-6 or -7 resulted in the production of heterodimers that were beneficial for bone formation as compared to BMP homodimers. Combination of BMP sequences could reduce the need for high BMP protein dosages and might enhance prolonged availability of the growth factors.

Local induction of inflammation affects bone formation.

To explore the influence of inflammatory processes on bone formation, we applied a new in vivo screening model. Confined biological pockets were first created in rabbits as a response to implanted bone cement discs. These biomembrane pockets were subsequently used to study the effects of inflammatory stimuli on ectopic bone formation within biphasic calcium phosphate (BCP) constructs loaded with TNF-α, lipopolysaccharide (LPS) or lipoteichoic acid (LTA), all with or without bone morphogenetic protein (BMP)-2. Analysis of bone formation after 12 weeks demonstrated that the inflammatory mediators were not bone-inductive in combination with the BCP alone, but inhibited or enhanced BMP-induced bone formation. LPS was associated with a strong inhibition of bone formation by BMP-2, while LTA and TNF-α showed a positive interaction with BMP-2. Since the biomembrane pockets did not interfere with bone formation and prevented the leakage of pro-inflammatory compounds to the surrounding tissue, the biomembrane model can be used for in vivo approaches to study local inflammation in conjunction with new bone formation. Using this model, it was shown that the modulation of the inflammatory response could be beneficial or detrimental to the subsequent bone formation process. The co-delivery of inflammatory factors and bone-related growth factors should be further explored as a strategy to enhance the bone-forming efficacy of bone substitutes.

Hyaluronic Acid-Based Hydrogel Coating Does Not Affect Bone Apposition at the Implant Surface in a Rabbit Model.

BACKGROUND: Uncemented orthopaedic implants rely on the bone-implant interface to provide stability, therefore it is essential that a coating does not interfere with the bone-forming processes occurring at the implant interface. In addition, local application of high concentrations of antibiotics for prophylaxis or treatment of infection may be toxic for osteoblasts and could impair bone growth. QUESTIONS/PURPOSES: In this animal study, we investigated the effect of a commercially available hydrogel, either unloaded or loaded with 2% vancomycin. We asked, does unloaded hydrogel or hydrogel with vancomycin (1) interfere with bone apposition and timing of bone deposition near the implant surface; and (2) induce a local or systemic inflammatory reaction as determined by inflammation around the implant and hematologic parameters. METHODS: In 18 New Zealand White rabbits, an uncoated titanium rod (n = 6), a rod coated with unloaded hydrogel (n = 6), or a rod coated with 2% vancomycin-loaded hydrogel (n = 6) was implanted in the intramedullary canal of the left tibia. After 28 days, the bone volume fraction near the implant was measured with microCT analysis, inflammation was semiquantitatively scored on histologic sections, and timing of bone apposition was followed by semiquantitative scoring of fluorochrome incorporation on histologic sections. Two observers, blinded to the treatment, scored the sections and reconciled their scores if there was a disagreement. The hematologic inflammatory reaction was analyzed by measuring total and differential leukocyte counts and erythrocyte sedimentation rates in blood. With group sizes of six animals per group, we had 79% power to detect a difference of 25% in histologic scoring for infection and inflammation. RESULTS: No differences were found in the amount of bone apposition near the implant in the No Gel group (48.65% ± 14.95%) compared with the Gel group (59.97% ± 5.02%; mean difference [MD], 11.32%; 95% CI, -3.89% to 26.53%; p = 0.16) or for the Van2 group (56.12% ± 10.06%; MD, 7.46; 95% CI, -7.75 to 22.67; p = 0.40), with the numbers available. In addition, the scores for timing of bone apposition did not differ between the No Gel group (0.50 ± 0.55) compared with the Gel group (0.33 ± 0.52; MD, -0.17; 95% CI, -0.86 to 0.53; p = 0.78) or the Van2 group (0.83 ± 0.41; MD, 0.33; 95% CI, -0.36 to 1.03; p = 0.42). Furthermore, we detected no differences in the histopathology scores for inflammation in the No Gel group (2.33 ± 1.67) compared with the Gel group (3.17 ± 1.59; MD, 0.83; 95% CI, -0.59 to 2.26; p = 0.31) or to the Van2 group (2.5 ± 1.24; MD, 0.17; 95% CI, -1.26 to 1.59; p = 0.95). Moreover, no differences in total leukocyte count, erythrocyte sedimentation rate, and neutrophil, monocyte, eosinophil, basophil, and lymphocyte counts were present between the No Gel or Van2 groups compared with the Gel control group, with the numbers available. CONCLUSION: The hydrogel coated on titanium implants, unloaded or loaded with 2% vancomycin, had no effect on the volume or timing of bone apposition near the implant, and did not induce an inflammatory reaction in vivo, with the numbers available. CLINICAL RELEVANCE: Antibiotic-loaded hydrogel may prove to be a valuable option to protect orthopaedic implants from bacterial colonization. Future clinical safety studies will need to provide more evidence that this product does not impair bone formation near the implant and prove the safety of this product.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics.

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone.

Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume.

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

Growth factor-induced osteogenesis in a novel radiolucent bone chamber.

Treatment of large bone defects is currently performed using mainly autograft or allograft bone. There are important drawbacks to bone grafting, such as limited availability, donor site morbidity in the case of autograft and inferior performance of allografts. Therefore, there is a great need for a suitable bone graft substitute. In order to evaluate efficiently newly developed biomaterials and factors intended for orthopaedic surgery, the bone chamber is a very suitable model. To allow longitudinal investigation of bone growth with µCT, a new bone chamber made of radiolucent polyether ether ketone (PEEK) was developed and studied for its feasibility. Therefore, PEEK bone chambers were placed on rat tibiae, and filled with vehicle (Matrigel without growth factors, negative controls), with bone morphogenetic protein 2 (BMP-2, positive controls), or a mix of growth factors combining BMP-2, vascular endothelial growth factor and the chemokine stromal cell-derived factor 1α, all laden on gelatin microspheres for controlled release (combined growth factors). Growth factor presence led to a significant increase in bone formation after 8 weeks, which subsided after 12 weeks, underlining the importance of longitudinal analysis. We conclude that the PEEK-bone chamber is a suitable translational animal model to assess orthotopic bone formation in a longitudinal manner.

Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee: a controlled laboratory and human cadaveric study.

OBJECTIVE: The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. METHODS: An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1 × 10(6) cells/ml fibrin glue (Tissucol, Baxter) in vitro. Depth-dependent cell viability was assessed for both methods with confocal microscopy. Constructs were cultured for 21 days to assess matrix production. A controlled human cadaveric study (n = 8) was performed to test the feasibility of the procedure in which defects were filled with either arthroscopic airbrushing or needle extrusion. All knees were subjected to 60 min of continuous passive motion and scored on outline attachment and defect filling. RESULTS: Spraying both chondrocytes and chondrons in fibrin glue resulted in a homogenous cell distribution throughout the scaffold. No difference in viability or matrix production between application methods was found nor between chondrons and chondrocytes. The cadaveric study revealed that airbrushing was highly feasible, and that defect filling through needle extrusion was more difficult to perform based on fibrin glue adhesion and gravity-induced seepage. Defect outline and coverage scores were consistently higher for extrusion, albeit not statistically significant. CONCLUSION: Both chondrons and chondrocytes can be evenly distributed in a sprayed fibrin glue scaffold without affecting viability while supporting matrix production. The airbrush technology is feasible, easier to perform than needle extrusion and allows for reproducible arthroscopic filling of cartilage defects.

Cartilage regeneration using zonal chondrocyte subpopulations: a promising approach or an overcomplicated strategy?

Cartilage defects heal imperfectly and osteoarthritic changes develop frequently as a result. Although the existence of specific behaviours of chondrocytes derived from various depth-related zones in vitro has been known for over 20 years, only a relatively small body of in vitro studies has been performed with zonal chondrocytes and current clinical treatment strategies do not reflect these native depth-dependent (zonal) differences. This is surprising since mimicking the zonal organization of articular cartilage in neo-tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft. Although some research groups including our own have made considerable progress in tailoring culture conditions using specific growth factors and biomechanical loading protocols, we conclude that an optimal regime has not yet been determined. Other unmet challenges include the lack of specific zonal cell sorting protocols and limited amounts of cells harvested per zone. As a result, the engineering of functional tissue has not yet been realized and no long-term in vivo studies using zonal chondrocytes have been described. This paper critically reviews the research performed to date and outlines our view of the potential future significance of zonal chondrocyte populations in regenerative approaches for the treatment of cartilage defects. Secondly, we briefly discuss the capabilities of additive manufacturing technologies that can not only create patient-specific grafts directly from medical imaging data sets but could also more accurately reproduce the complex 3D zonal extracellular matrix architecture using techniques such as hydrogel-based cell printing.

Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site.

OBJECTIVE: Interactions between chondrocytes and their native pericellular matrix provide optimal circumstances for regeneration of cartilage. However, cartilage diseases such as osteoarthritis change the pericellular matrix, causing doubt to them as a cell source for autologous cell therapy. METHODS: Chondrons and chondrocytes were isolated from stifle joints of goats in which cartilage damage was surgically induced in the right knee. After 4 weeks of regeneration culture, DNA content and proteoglycan and collagen content and release were determined. RESULTS: The cartilage regenerated by chondrons isolated from the damaged joint contained less proteoglycans and collagen compared to chondrons from the same harvest site in the nonoperated knee (P < 0.01). Besides, chondrons still reflected whether they were isolated from a damaged joint, even if they where isolated from the opposing or adjacent condyle. Although chondrocytes did not reflect this diseased status of the joint, chondrons always outperformed chondrocytes, even when isolated from the damaged joints (P < 0.0001). Besides increased cartilage production, the chondrons showed less collagenase activity compared to the chondrocytes. CONCLUSION: Chondrons still outperform chondrocytes when they were isolated from a damaged joint and they might be a superior cell source for articular cartilage repair and cell-induced cartilage formation.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes.

OBJECTIVE: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN: OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. RESULTS: Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. CONCLUSION: Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

Clinical outcome after traumatic spinal fractures in patients with ankylosing spinal disorders compared with control patients.

BACKGROUND CONTEXT: The clinical outcome of patients with ankylosing spinal disorders (ASDs) sustaining a spinal fracture has been described to be worse compared with the general trauma population. PURPOSE: To investigate clinical outcome (neurologic deficits, complications, and mortality) after spinal injury in patients with ankylosing spondylitis (AS) and diffuse idiopathic skeletal hyperostosis (DISH) compared with control patients. STUDY DESIGN: Retrospective cohort study. PATIENT SAMPLE: All patients older than 50 years and admitted with a traumatic spinal fracture to the Emergency Department of the University Medical Center Utrecht, the Netherlands, a regional level-1 trauma center and tertiary referral spine center. OUTCOME MEASURES: Data on comorbidity (Charlson comorbidity score), mechanism of trauma, fracture characteristics, neurologic deficit, complications, and in-hospital mortality were collected from medical records. METHODS: With logistic regression analysis, the association between the presence of an ASD and mortality was investigated in relation to other known risk factors for mortality. RESULTS: A total of 165 patients met the inclusion criteria; 14 patients were diagnosed with AS (8.5%), 40 patients had DISH (24.2%), and 111 patients were control patients (67.3%). Ankylosing spinal disorder patients were approximately five years older than control patients and predominantly of male gender. The Charlson comorbidity score did not significantly differ among the groups, but Type 2 diabetes mellitus and obesity were more prevalent among DISH patients. In many AS and DISH cases, fractures resulted from low-energy trauma and showed a hyperextension configuration. Patients with AS and DISH were frequently admitted with a neurologic deficit (57.1% and 30.0%, respectively) compared with controls (12.6%; p=.002), which did not improve in the majority of cases. In AS and DISH patients, complication and mortality rates were significantly higher than in controls. Logistic regression analysis showed the parameters age and presence of DISH to be independently, statistically significantly related to mortality. CONCLUSIONS: Many patients with AS and DISH showed unstable (hyperextension) fracture configurations and neurologic deficits. Complication and mortality rates were higher in patients with ASD compared with control patients. Increasing age and presence of DISH are predictors of mortality after a spinal fracture.

Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.

OBJECTIVE: Although extracellular matrix (ECM)-derived scaffolds have been extensively studied and applied in a number of clinical applications, the use of ECM as a biomaterial for (osteo)chondral regeneration is less extensively explored. This study aimed at evaluating the chondrogenic potential of cells seeded on cartilage-derived matrix (CDM) scaffolds in vitro. DESIGN: Scaffolds were generated from decellularized equine articular cartilage and seeded with either chondrocytes or multipotent stromal cells (MSCs). After 2, 4, and 6 weeks of in vitro culture, CDM constructs were analyzed both histologically (hematoxylin and eosin, Safranin-O, collagen types I and II) and biochemically (glycosaminoglycan [GAG] and DNA content). RESULTS: After 4 weeks, both cell types demonstrated chondrogenic differentiation; however, the MSCs significantly outperformed chondrocytes in producing new GAG-containing cartilaginous matrix. CONCLUSION: These promising in vitro results underscore the potency of CDM scaffolds in (osteo)chondral defect repair.

A validated new histological classification for intervertebral disc degeneration.

UNLABELLED: Histology is an important outcome variable in basic science and pre-clinical studies regarding intervertebral disc degeneration (IVD). Nevertheless, an adequately validated histological classification for IVD degeneration is still lacking and the existing classifications are difficult to use for inexperienced observers. OBJECTIVE: Therefore the aim of this study was to develop and to validate a new histological classification for IVD degeneration. Moreover, the new classification was compared to the frequently used non-validated classification. METHODS: The new classification was applied to human IVD sections. The sections were scored twice by two independent inexperienced observers, twice by two experienced IVD researchers and once by a pathologist. For comparison, the sections were also scored according to the classification described by Boos et al. by two experienced IVD researchers. Macroscopic grading according Thompson et al., glycosaminoglycan (GAG) content and age were used for validation. RESULTS: The new classification had an excellent intra- and a good inter-observer reliability. Intraclass Correlation Coefficients (ICC) were 0.83 and 0.74, respectively. Intra- and inter-observer reliability were comparable for experienced and inexperienced observers. Statistically significant correlations were found between the new classification, macroscopic score, GAG content in the nucleus pulposus (NP) and age; Correlation coefficient (CC) 0.79, -0.62 and 0.68, respectively. The CCs of the Boos classification were all lower compared to the new classification. CONCLUSION: the new histological classification for IVD degeneration is a valid instrument for evaluating IVD degeneration in human IVD sections and is suitable for inexperienced and experienced researchers.

One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats.

OBJECTIVE: The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work already showed a positive stimulation of cartilage specific matrix production when chondrocytes and MNF cells were combined. Therefore, this study aimed to evaluate cartilage regeneration using a one-stage procedure combining MNF cells and primary chondrocytes for the treatment of focal cartilage lesions in goats compared to microfracture treatment. DESIGN: Freshly created focal cartilage defects were treated with either a combination of chondrocytes and MNF cells embedded in fibrin glue or microfracture treatment. After 6 months follow-up local regeneration as well as the general joint cartilage health were evaluated using validated scores and biochemical assays. RESULTS: Macroscopic (P = 0.015) scores for the cartilage surface at the treated defect were, after 6 months, significantly higher for the chondrocyteMNF treatment compared to microfracture-treated defects, but microscopic scores were not (P = 0.067). The articulating cartilage showed more (P = 0.005) degeneration following microfracture treatment compared to chondrocyteMNF treatment. Biochemical glycosaminoglycans (GAG) evaluation did not reveal differences between the treatments. Both treatments had resulted in a slight to moderate cartilage degeneration at other locations in the joint. CONCLUSION: In conclusion, treatment of focal articular cartilage lesions in goats using a combination of MNF cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture, however needs further fine-tuning to decrease the negative influence on other joint compartments.

Delayed gadolinium enhanced MRI of cartilage (dGEMRIC) can be effectively applied for longitudinal cohort evaluation of articular cartilage regeneration.

OBJECTIVE: Delayed gadolinium enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) facilitates non-invasive evaluation of the glycosaminoglycan content in articular cartilage. The primary aim of this study was to show that the dGEMRIC technique is able to monitor cartilage repair following regenerative cartilage treatment. DESIGN: Thirty-one patients with a focal cartilage lesion underwent a dGEMRIC scan prior to cartilage repair surgery and at 3 and 12 months follow-up. At similar time points clinical improvement was monitored using the Knee injury and Osteoarthritis Outcome Score (KOOS) and Lysholm questionnaires. Per MRI scan several regions-of-interest (ROIs) were defined for different locations in the joint. The dGEMRIC index (T1gd) was calculated for each ROI. Repeated-measures analysis of variance (RMANOVA) analysis was used to evaluate improvement in clinical scores and MRI T1gd over time. Also regression analysis was performed to show the influence of local repair on cartilage quality at distant locations in the knee. RESULTS: Clinical scores and the dGEMRIC T1gd per ROI showed a statistically significant improvement (P < 0.01), from baseline, at 12 months follow-up. Also, improvement from baseline in T1gd of the ROI defining the treated cartilage defect showed a direct relationship (P < 0.007) to the improvement of the T1gd of ROI at other locations in the joint. CONCLUSIONS: The dGEMRIC MRI protocol is a useful method to evaluate cartilage repair. In addition, local cartilage repair influenced the cartilage quality at other location in the joint. These findings validate the use of dGEMRIC for non-invasive evaluation of the effects of cartilage regeneration.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles.

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.