BMP-2 gene delivery in cell-loaded and cell-free constructs for bone regeneration.

To induce osteogenicity in bone graft substitutes, plasmid-based expression of BMP-2 (pBMP-2) has been successfully applied in gene activated matrices based on alginate polymer constructs. Here, we investigated whether cell seeding is necessary for non-viral BMP-2 gene expression in vivo. Furthermore, to gain insight in the role of BMP-producing cells, we compared inclusion of bone progenitor cells with non-osteogenic target cells in gene delivery constructs. Plasmid DNA encoding GFP (pGFP) was used to trace transfection of host tissue cells and seeded cells in a rat model. Transgene expression was followed in both cell-free alginate-ceramic constructs as well as constructs seeded with syngeneic fibroblasts or multipotent mesenchymal stromal cells (MSCs). Titration of pGFP revealed that the highest pGFP dose resulted in frequent presence of positive host cells in the constructs. Both cell-loaded groups were associated with transgene expression, most effectively in the MSC-loaded constructs. Subsequently, we investigated effectiveness of cell-free and cell-loaded alginate-ceramic constructs with pBMP-2 to induce bone formation. Local BMP-2 production was found in all groups containing BMP-2 plasmid DNA, and was most pronounced in the groups with MSCs transfected with high concentration pBMP-2. Bone formation was only apparent in the recombinant protein BMP-2 group. In conclusion, we show that non-viral gene delivery of BMP-2 is a potentially effective way to induce transgene expression in vivo, both in cell-seeded as well as cell-free conditions. However, alginate-based gene delivery of BMP-2 to host cells or seeded cells did not result in protein levels adequate for bone formation in this setting, calling for more reliable scaffold compatible transfection methods.

A Human Hematopoietic Niche Model Supporting Hematopoietic Stem and Progenitor Cells In Vitro.

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk, a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability, clonogenic hematopoietic potential, and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells, together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel, the supportive cells self-assemble into a hypoxic stromal network, stimulating CD34+ CD38+ cell formation, while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks, in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly, the primary in vitro niche model supports HSPCs with no cytokine addition. Overall, the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches, in the context of normal hematopoiesis or blood-related diseases.

PTH decreases in vitro human cartilage regeneration without affecting hypertrophic differentiation.

Regenerated cartilage formed after Autologous Chondrocyte Implantation may be of suboptimal quality due to postulated hypertrophic changes. Parathyroid hormone-related peptide, containing the parathyroid hormone sequence (PTHrP 1-34), enhances cartilage growth during development and inhibits hypertrophic differentiation of mesenchymal stromal cells (MSCs) and growth plate chondrocytes. This study aims to determine the possible anabolic and/or hypertrophic effect of PTH on human articular chondrocytes. Healthy human articular cartilage-derived chondrocytes (n = 6 donors) were cultured on type II collagen-coated transwells with/without 0.1 or 1.0 µM PTH from day 0, 9, or 21 until the end of culture (day 28). Extracellular matrix production, (pre)hypertrophy and PTH signaling were assessed by RT-qPCR and/or immunohistochemistry for collagen type I, II, X, RUNX2, MMP13, PTHR1 and IHH and by determining glycosaminoglycan production and DNA content. The Bern score assessed cartilage quality by histology. Regardless of the concentration and initiation of supplementation, PTH treatment significantly decreased DNA and glycosaminoglycan content and reduced the Bern score compared with controls. Type I collagen deposition was increased, whereas PTHR1 expression and type II collagen deposition were decreased by PTH supplementation. Expression of the (pre)hypertrophic markers MMP13, RUNX2, IHH and type X collagen were not affected by PTH. In conclusion, PTH supplementation to healthy human articular chondrocytes did not affect hypertrophic differentiation, but negatively influenced cartilage quality, the tissues' extracellular matrix and cell content. Although PTH may be an effective inhibitor of hypertrophic differentiation in MSC-based cartilage repair, care may be warranted in applying accessory PTH treatment due to its effects on articular chondrocytes.

Endosteal and Perivascular Subniches in a 3D Bone Marrow Model for Multiple Myeloma.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138+ myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138+ myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.

Effect of different sustained bone morphogenetic protein-2 release kinetics on bone formation in poly(propylene fumarate) scaffolds.

To investigate the effect of sustained bone morphogenetic protein-2 (BMP-2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic-co-glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP-2 release by varying the lactic-to-glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP-2 release kinetics were determined by analyzing radiolabeled 125 I-BMP-2. Biological activity of released BMP-2 was tested using a W20-17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP-2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP-2 per time point in the first weeks. Micro-CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP-2 sustained release had no significant effect on BMP-2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477-487, 2018.

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity.

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration.

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.

OP-1 Compared with Iliac Crest Autograft in Instrumented Posterolateral Fusion: A Randomized, Multicenter Non-Inferiority Trial.

BACKGROUND: Spinal fusion with the use of autograft is a commonly performed procedure. However, harvesting of bone from the iliac crest is associated with complications. Bone morphogenetic proteins (BMPs) are extensively used as alternatives, often without sufficient evidence of safety and efficacy. The purpose of this study was to investigate non-inferiority of osteogenic protein-1 (OP-1, also known as BMP-7) in comparison with iliac crest bone graft in posterolateral fusions. METHODS: This study was a randomized, controlled multicenter trial. Patients who underwent a single-level instrumented posterolateral fusion of the lumbar spine for degenerative or isthmic spondylolisthesis with symptoms of neurological compression were randomized to receive OP-1 combined with local bone (OP-1 group) or autologous bone graft from the iliac crest combined with local bone (autograft group). The primary outcome was overall success, defined as a combination of clinical success and evidence of fusion on computed tomography (CT) scans, at one year postoperatively. RESULTS: One hundred and nineteen patients were included in the study, and analysis of the overall outcome was performed for 113. Non-inferiority of OP-1 compared with iliac crest autograft was not found at one year, with a success rate of 40% in the OP-1 group versus 54% in the autograft group (risk difference = -13.3%, 90% confidence interval [CI] = -28.6% to +2.10%). This was due to the lower rate of fusion (the primary aim of OP-1 application) seen on the CT scans in the OP-1 group (54% versus 74% in the autograft group, p = 0.03). There were no adverse events that could be directly related to the use of OP-1. CONCLUSIONS: OP-1 with a collagen carrier was not as effective as autologous iliac crest bone for achieving fusion and cannot be recommended in instrumented posterolateral lumbar fusion procedures. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Proinflammatory T cells and IL-17 stimulate osteoblast differentiation.

The local immune response is important to consider when the aim is to improve bone regeneration. Recently T lymphocytes and their associated cytokines have been identified as regulators in fracture callus formation, but it is not known whether T cells affect bone progenitor cells directly. The goal of this in vitro study was to investigate the role of different T cell subsets and their secreted factors on the osteogenic differentiation of human mesenchymal stem cells (MSCs). Significant increases in the alkaline phosphatase activity and the subsequent matrix mineralization by MSCs were found after their exposure to activated T cells or activated T cell-derived conditioned medium. Blocking IFN-γ in the conditioned medium abolished its pro-osteogenic effect, while blocking TGF-ß further enhanced osteogenesis. The relative contribution of an anti- or proinflammatory T cell phenotype in MSC osteogenic differentiation was studied next. Enrichment of the fraction of anti-inflammatory regulatory T cells had no beneficial osteogenic effect. In contrast, soluble factors derived from enriched T helper 17 cells upregulated the expression of osteogenic markers by MSCs. IL-17A, and IL-17F, their main proinflammatory cytokines, similarly exhibited strong osteogenic effects when exposed directly to MSCs. IL-17A in particular showed a synergistic action together with bone morphogenetic protein 2. These results indicate that individual T cell subsets, following their activation, affect osteoblast maturation in a different manner through the production of soluble factors. From all T cells, the proinflammatory T cells, including the T helper 17 cells, are most stimulatory for osteogenesis.

Missed low-grade infection in suspected aseptic loosening has no consequences for the survival of total hip arthroplasty.

BACKGROUND AND PURPOSE: Aseptic loosening and infection are 2 of the most common causes of revision of hip implants. Antibiotic prophylaxis reduces not only the rate of revision due to infection but also the rate of revision due to aseptic loosening. This suggests under-diagnosis of infections in patients with presumed aseptic loosening and indicates that current diagnostic tools are suboptimal. In a previous multicenter study on 176 patients undergoing revision of a total hip arthroplasty due to presumed aseptic loosening, optimized diagnostics revealed that 4-13% of the patients had a low-grade infection. These infections were not treated as such, and in the current follow-up study the effect on mid- to long-term implant survival was investigated. PATIENTS AND METHODS: Patients were sent a 2-part questionnaire. Part A requested information about possible re-revisions of their total hip arthroplasty. Part B consisted of 3 patient-related outcome measure questionnaires (EQ5D, Oxford hip score, and visual analog scale for pain). Additional information was retrieved from the medical records. The group of patients found to have a low-grade infection was compared to those with aseptic loosening. RESULTS: 173 of 176 patients from the original study were included. In the follow-up time between the revision surgery and the current study (mean 7.5 years), 31 patients had died. No statistically significant difference in the number of re-revisions was found between the infection group (2 out of 21) and the aseptic loosening group (13 out of 152); nor was there any significant difference in the time to re-revision. Quality of life, function, and pain were similar between the groups, but only 99 (57%) of the patients returned part B. INTERPRETATION: Under-diagnosis of low-grade infection in conjunction with presumed aseptic revision of total hip arthroplasty may not affect implant survival.

Proinflammatory Mediators Enhance the Osteogenesis of Human Mesenchymal Stem Cells after Lineage Commitment.

Several inflammatory processes underlie excessive bone formation, including chronic inflammation of the spine, acute infections, or periarticular ossifications after trauma. This suggests that local factors in these conditions have osteogenic properties. Mesenchymal stem cells (MSCs) and their differentiated progeny contribute to bone healing by synthesizing extracellular matrix and inducing mineralization. Due to the variation in experimental designs used in vitro, there is controversy about the osteogenic potential of proinflammatory factors on MSCs. Our goal was to determine the specific conditions allowing the pro-osteogenic effects of distinct inflammatory stimuli. Human bone marrow MSCs were exposed to tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). Cells were cultured in growth medium or osteogenic differentiation medium. Alternatively, bone morphogenetic protein 2 (BMP-2) was used as osteogenic supplement to simulate the conditions in vivo. Alkaline phosphatase activity and calcium deposition were indicators of osteogenicity. To elucidate lineage commitment-dependent effects, MSCs were pre-differentiated prior treatment. Our results show that TNF-α and LPS do not affect the expression of osteogenic markers by MSCs in the absence of an osteogenic supplement. In osteogenic differentiation medium or together with BMP-2 however, these mediators highly stimulated their alkaline phosphatase activity and subsequent matrix mineralization. In pre-osteoblasts, matrix mineralization was significantly increased by these mediators, but irrespective of the culture conditions. Our study shows that inflammatory factors potently enhance the osteogenic capacity of MSCs. These properties may be harnessed in bone regenerative strategies. Importantly, the commitment of MSCs to the osteogenic lineage greatly enhances their responsiveness to inflammatory signals.

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.

Reconsidering the ethics of sham interventions in an era of emerging technologies.

BACKGROUND: Our aim was to ethically evaluate the arguments in favor and against sham interventions, as presented in literature. Two developments underscore the need to reconsider the ethics of sham interventions. First, the number of clinical trials investigating interventions in the field of regenerative medicine are increasing, in which the choice for a placebo requires an invasive intervention. Second, the increased awareness of the lack of systematic research in surgery stresses the need to discuss the necessity and acceptability of sham-controlled clinical trials. METHODS: A systematic search in Medline was performed, of which 104 articles were considered relevant. RESULTS: Arguments in favor of a sham controlled design are that it increases the scientific validity and the benefits to society while at the same time the risks and harm can be acceptable. Arguments against sham controls include that they pose unacceptable risks to participants, present difficulties with informed consent, that the use of deceptive tactics is unethical, and that the feasibility of such controls is compromised because of a lack of public support. CONCLUSION: None of the published literature fully rejects sham interventions, and many regard sham interventions acceptable provided the conditions of scientific necessity, reasonable risks, and valid informed consent are fulfilled. Further debate should no longer address whether a sham control is ethically acceptable but rather when these conditions are fulfilled.

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.

Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles.

The natural process of endochondral bone formation in the growing skeletal system is increasingly inspiring the field of bone tissue engineering. However, in order to create relevant-size bone grafts, a cell carrier is required that ensures a high diffusion rate and facilitates matrix formation, balanced by its degradation. Therefore, we set out to engineer endochondral bone in gelatin methacrylamide (GelMA) hydrogels with embedded multipotent stromal cells (MSCs) and cartilage-derived matrix (CDM) particles. CDM particles were found to stimulate the formation of a cartilage template by MSCs in the GelMA hydrogel in vitro. In a subcutaneous rat model, this template was subsequently remodeled into mineralized bone tissue, including bone-marrow cavities. The GelMA was almost fully degraded during this process. There was no significant difference in the degree of calcification in GelMA with or without CDM particles: 42.5 ± 2.5% vs. 39.5 ± 8.3% (mean ± standard deviation), respectively. Interestingly, in an osteochondral setting, the presence of chondrocytes in one half of the constructs fully impeded bone formation in the other half by MSCs. This work offers a new avenue for the engineering of relevant-size bone grafts, by the formation of endochondral bone within a degradable hydrogel.

Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration.

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

Clinical and radiological results 6 years after treatment of traumatic thoracolumbar burst fractures with pedicle screw instrumentation and balloon assisted endplate reduction.

BACKGROUND CONTEXT: When used to fixate traumatic thoracolumbar burst fractures, pedicle screw constructs may fail in the presence of severe vertebral body comminution as the intervertebral disc can creep through the fractured endplates leading to insufficient anterior column support. Balloon-assisted endplate reduction (BAER) and subsequent calcium phosphate cement augmentation may prevent this event by restoring the disc space boundaries. The results of the first studies using BAER after pedicle screw fixation are encouraging, showing good fracture reduction, few complications, and minimal loss of correction at 2 years of follow-up. PURPOSE: To present the clinical and radiological outcome of 20 patients treated for traumatic thoracolumbar burst fractures with pedicle screws and BAER after a minimum of 6 years follow-up. STUDY DESIGN: Prospective trial. PATIENT SAMPLE: Twenty consecutive neurologically intact adult patients with traumatic thoracolumbar burst fractures were included. OUTCOME MEASURES: Radiological parameters (wedge/Cobb angle on plain radiographs and mid-sagittal anterior/central vertebral body height on magnetic resonance imaging scans) and patient reported parameters (EQ-5D and Oswestry Disability Index) were used. METHODS: All patients had previously undergone pedicle screw fixation and BAER with calcium phosphate cement augmentation. The posterior instrumentation was removed approximately 1.5 years after index surgery. Radiographs were obtained preoperatively, postoperatively, after removal of the pedicle screws, and at final follow-up (minimum 6 years post-trauma). Magnetic resonance imaging scans were obtained preoperatively, 1 month after index surgery, and 1 month after pedicle screw removal. Health questionnaires were filled out during the last outpatient visit. RESULTS: The pedicle screw instrumentation was removed uneventfully in all patients and posterolateral fusion was observed in every case. The mean wedge and Cobb angle converged to almost identical values (5.3° and 5.8°, respectively) and the mid-sagittal anterior and central endplates were reduced to approximately 90% and 80% of the estimated preinjury vertebral body height, respectively; this reduction was sustained at follow-up. Patient-reported outcomes showed favorable results in 79% of the patients. One patient required (posterior) reoperation due to adjacent osteoporotic vertebral body collapse after pedicle screw removal. CONCLUSIONS: Balloon-assisted endplate reduction is a safe and low-demanding adjunct to pedicle screw fixation for the treatment of traumatic thoracolumbar burst fractures. It may help achieve minimal residual deformity and reduce the number of secondary (anterior) procedures. Despite these positive findings, one in five patients experienced daily discomfort and disability.

Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes.

INTRODUCTION: This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue. METHODS: Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1ß, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF). RESULTS: In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤ 0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P < 0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤ 0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P < 0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P < 0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes. CONCLUSIONS: Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

CXCL12/stromal-cell-derived factor-1 effectively replaces endothelial progenitor cells to induce vascularized ectopic bone.

Bone defect healing is highly dependent on the simultaneous stimulation of osteogenesis and vascularization. In bone regenerative strategies, combined seeding of multipotent stromal cells (MSCs) and endothelial progenitor cells (EPCs) proves their mutual stimulatory effects. Here, we investigated whether stromal-cell-derived factor-1α (SDF-1α) stimulates vascularization by EPCs and whether SDF-1α could replace seeded cells in ectopic bone formation. Late EPCs of goat origin were characterized for their endothelial phenotype and showed to be responsive to SDF-1α in in vitro migration assays. Subsequently, subcutaneous implantation of Matrigel plugs that contained both EPCs and SDF-1α showed more tubule formation than constructs containing either EPCs or SDF-1α. Addition of either EPCs or SDF-1α to MSC-based constructs showed even more elaborate vascular networks after 1 week in vivo, with SDF-1α/MSC-laden groups showing more prominent interconnected networks than EPC/MSC-laden groups. The presence of abundant mouse-specific CD31/PECAM expression in these constructs confirmed ingrowth of murine vessels and discriminated between angiogenesis and vessel networks formed by seeded goat cells. Importantly, implantation of EPC/MSC or SDF-1α/MSC constructs resulted in indistinguishable ectopic bone formation. In both groups, bone onset was apparent at week 3 of implantation. Taken together, we demonstrated that SDF-1α stimulated the migration of EPCs in vitro and vascularization in vivo. Further, SDF-1α addition was as effective as EPCs in inducing the formation of vascularized ectopic bone based on MSC-seeded constructs, suggesting a cell-replacement role for SDF-1α. These results hold promise for the design of larger centimeter-scale, cell-free vascular bone grafts.