Stefan Schaeren

Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering.

Mesenchymal stem/stromal cells (MSC) are typically used to generate bone tissue by a process resembling intramembranous ossification, i.e., by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e., via remodeling of hypertrophic cartilaginous templates. To date, endochondral bone formation has not been reproduced using human, clinically compliant cell sources. Here, we aimed at engineering tissues from bone marrow-derived, adult human MSC with an intrinsic capacity to undergo endochondral ossification. By analogy to embryonic limb development, we hypothesized that successful execution of the endochondral program depends on the initial formation of hypertrophic cartilaginous templates. Human MSC, subcutaneously implanted into nude mice at various stages of chondrogenic differentiation, formed bone trabeculae only when they had developed in vitro hypertrophic tissue structures. Advanced maturation in vitro resulted in accelerated formation of larger bony tissues. The underlying morphogenetic process was structurally and molecularly similar to the temporal and spatial progression of limb bone development in embryos. In particular, Indian hedgehog signaling was activated at early stages and required for the in vitro formation of hypertrophic cartilage. Subsequent development of a bony collar in vivo was followed by vascularization, osteoclastic resorption of the cartilage template, and appearance of hematopoietic foci. This study reveals the capacity of human MSC to generate bone tissue via an endochondral program and provides a valid model to study mechanisms governing bone development. Most importantly, this process could generate advanced grafts for bone regeneration by invoking a “developmental engineering” paradigm.

Dynamic stabilization in addition to decompression for lumbar spinal stenosis with degenerative spondylolisthesis

Study Design. Prospective clinical study. Objective. To test whether elastic stabilization with the Dynesys system (Zimmer Spine, Minneapolis, MN) provides enough stability to prevent further progression of spondylolisthesis as well as instability after decompression for spinal stenosis with degenerative spondylolisthesis. Summary of Background Data. In spinal stenosis with degenerative spondylolisthesis, decompression and fusion is widely recommended. However, patients have donor site pain. In 1994, a dynamic transpedicular system (Dynesys) was introduced to the market, stating that stabilization is possible without bone grafting. Methods. A total of 26 patients (mean age 71 years) with lumbar spinal stenosis and degenerative spondylolisthesis underwent interlaminar decompression and dynamic stabilization with the Dynesys system. Minimum follow-up was 2 years. Operative data, clinical outcome, and plain and flexion/extension radiographs were obtained and compared to preoperative and postoperative data. Results. Mean leg pain decreased significantly (P < 0.01), and mean walking distance improved significantly to more than 1000 m (P < 0.01). There were 5 patients (21%) who still had some claudication. A total of 21 patients (87.5%) would undergo the same procedure again. Radiographically, no significant progression of spondylolisthesis could be detected. The implant failure rate was 17%, and none of them were clinically symptomatic. Conclusions. In elderly patients with spinal stenosis with degenerative spondylolisthesis, dynamic stabilization with the Dynesys system in addition to decompression leads to similar clinical results as seen in established protocols using decompression and fusion with pedicle screws. It maintains enough stability to prevent further progression of spondylolisthesis or instability. With the Dynesys system, no bone grafting is necessary, therefore, donor site morbidity can be avoided.

Minimum four-year follow-up of spinal stenosis with degenerative spondylolisthesis treated with decompression and dynamic stabilization.

Study Design. Prospective clinical study. Objective. To test whether posterior dynamic stabilization in situ with Dynesys (Zimmer Spine, Minneapolis, MN) can maintain enough stability to prevent progression of spondylolisthesis in long-term follow-up. Summary of Background Data. In spinal stenosis with degenerative spondylolisthesis, decompression and fusion are widely recommended. However, drawbacks of fusion remain length of surgery, blood loss, possible adjacent segment disease, errant instrumentation, nonunion, and pain at the bone donor site. The Dynesys system was introduced to stabilize the spine without adding bone graft for fusion. Excellent 2 years results have been reported. Methods. Twenty-six consecutive patients (mean age, 71 years) with symptomatic lumbar spinal stenosis and degenerative spondylolisthesis underwent interlaminar decompression and stabilization with Dynesys. Patients were evaluated clinically and radiologically after a minimum follow-up of 4 years. Results. Nineteen of 26 patients could be evaluated with a mean follow-up of 52 months (range, 48–57 months). Pain on VAS and walking distance improved significantly (P < 0.001) at 2 years and remained unchanged at 4 years follow-up. Radiographically, spondylolisthesis did not progress and the motion segments remained stable, even in the 3 patients who showed slight screw-loosening at 2 and 4 years follow-up. One patient showed screw breakage with low back pain and motion at the instrumented level in flexion/extension views. At 4 years follow-up, 47% of the patients showed some degeneration at adjacent levels. Overall, patient satisfaction remained high as 95% would undergo the same procedure again. Conclusion. In elderly patients with spinal stenosis and degenerative spondylolisthesis, decompression and dynamic stabilization lead to excellent clinical and radiologic results. It maintains enough stability to prevent progression of spondylolisthesis. Because no bone grafting is necessary, donor site morbidity, which is one of the main drawbacks of fusion is eliminated. However, the degenerative disease still is progressive and degeneration at adjacent motion segments remains a problem.

Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells

OBJECTIVE To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications. METHODS Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), human platelet lysate, or interferon-γ (IFNγ). The roles of cell proliferation and growth factor-induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity. RESULTS FGF-2 and, to a lesser extent, PDGF-BB induced in adult human MSCs the expression of HLA-DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA-DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA-DR expression in human MSCs. HLA-DR expression occurred following FGF-specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt controlled cell proliferation and HLA-DR expression, but only MAPK/ERK-1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA-DR transcription. CONCLUSION The induction of functional HLA-DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

In vitro osteogenic differentiation and in vivo bone-forming capacity of human isogenic jaw periosteal cells and bone marrow stromal cells.

Objective:To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. Summary Background Data:JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. Methods:BMSCs and JPCs from the same donors (N = 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. Results:JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. Conclusions:Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo.

Assessment of Nerve Damage Using a Novel Ultrasonic Device for Bone Cutting

f m ( l a S f t v f t n oral, maxillofacial, and spinal surgery, osteotomy ften must be performed in close vicinity to nerve issue, with the potential risk of transient or permaent neurologic injuries (eg, to trigeminal nerve ranches). Traditional tools, such as rotating burs nd oscillating saws, are highly effective in cutting one tissue but are not selective for bone, and thus an produce significant harm to surrounding soft tisues, especially nerves. Piezosurgery (Mebiotec, Sestri Levante, Italy), a deice for bone-cutting based on low-frequency (25 to 9 kHz) ultrasonic vibrations, has recently been introuced in oral and maxillofacial surgery. This device an improve the control and precision of osteotomy nd improve bone healing due to reduced local rauma. A recent in vitro study of the use of Piezourgery for transposition of the inferior alveolar nerve n the mandibles of cadaver sheep showed that this echnique caused roughening of the epineurium withut affecting deeper structures and induced less inury than a conventional rotary bur. A pilot clinical tudy found that Piezosurgery reduced neurosensory isturbances in orthognathic surgery of the mandile.

Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c.

Bone marrow (BM) mesenchymal stem/stromal cells (MSC) are a heterogeneous population of multipotent progenitors currently under investigation for a variety of applications in regenerative medicine. While self‐renewal of stem cells in different tissues has been demonstrated to be regulated by specialized microenvironments called niches, it is still unclear whether a self‐renewing niche also exists for MSC. Here, we show that primary human BM cultures contain a population of intrinsically non‐adherent mesenchymal progenitors (NAMP) with features of more primitive progenitors than the initially adhering colony‐forming units‐fibroblast (CFU‐f). In fact, NAMP could generate an adherent progeny: (a) enriched with early mesenchymal populations (CD146+, SSEA‐1+, and SSEA‐4+); (b) with significantly greater proliferation and multilineage differentiation potential in vitro; and (c) capable of threefold greater bone formation in vivo than the corresponding CFU‐f. Upon serial replating, NAMP were able to regenerate and expand in suspension as non‐adherent clonogenic progenitors, while also giving rise to an adherent progeny. This took place at the cost of a gradual loss of proliferative potential, shown by a reduction in colony size, which could be completely prevented when NAMP were expanded on the initially adhering BM fraction. Mechanistically, we found that NAMP crucially depend on fibroblast growth factor (FGF)‐2 signaling through FGFR2c for their survival and expansion. Furthermore, NAMP maintenance depends at least in part on humoral signals distinct from FGF‐2. In conclusion, our data show a niche/progenitor organization in vitro, in which the BM adherent fraction provides a self‐renewing microenvironment for primitive NAMP. Stem Cells2012;30:1455–1464

Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15

Objectives: To investigate whether human bone marrow-derived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor γ-chain (γc) in the absence of T cell receptor (TCR) triggering. Methods: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by 3H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. Results: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell–cell contact. Conclusions: There is a dichotomous effect of MSCs/ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs.

Implant removal after posterior stabilization of the thoraco-lumbar spine

IntroductionImplant removal because of pain after posterior fusion in the thoracic and lumbar spine is a widely performed operation. We conducted a retrospective study to examine whether patients benefit from implant removal.Patients and methods57 patients (29 males, 28 females, mean age 46.5 years) who have undergone removal of pedicle screws because of pain and discomfort were interviewed 6–24 months postoperatively. Fracture was the initial diagnosis in 40% of the patients and degenerative spine disease in 58%. The following factors were evaluated: patient satisfaction and postoperative outcome, patients’ native language and psychological background, operative data, hospital stay and complications.ResultsPain decreased significantly from 62 to 48 on visual analogue scale postoperatively. Complications occurred in five patients (8.8%). 36 patients (61%) stated they had some benefit from the operation, but only seven patients (12%) were free of pain completely. 36 patients (63%) would undergo the same procedure again. Outcome in the subgroup of foreigners was significantly worse, though the psychological background did not affect the outcome. Preoperative diagnostic infiltration was helpful in 9 of 13 patients.ConclusionRemoval of pedicle screws because of back pain may be effective, but complete remission of symptoms could be achieved in only 12% of patients. However, 63% of patients would undergo hardware removal again. Preoperative diagnostic infiltration can help to predict the outcome but results are inconsistent. Communication difficulties may worsen the outcome. Surgeons should consider these results when planning implant removal and patients should be informed thoroughly to avoid too high expectations.

Differentiation-dependent up-regulation of BMP-2, TGF-β1, and VEGF expression by FGF-2 in human bone marrow stromal cells

Background: Bone tissue formation by bone marrow stromal cells may be supported and enhanced by multiple growth factors, particularly in cases of a compromised local microenvironment. In this study, the authors hypothesized that fibroblast growth factor (FGF)-2 can stimulate the production by human bone marrow stromal cells of osteogenic [i.e., bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-β1] and angiogenic [i.e., vascular endothelial growth factor (VEGF)] factors. Methods: Human bone marrow stromal cells from six donors were expanded for two passages (expansion phase) and subsequently cultivated in osteogenic medium containing ascorbic acid, β-glycerophosphate, and dexamethasone (differentiation phase). After each phase, cells were transferred into serum-free medium with or without FGF-2 at different concentrations and for different times, and the expression of BMP-2, TGF-β1, and VEGF was quantified at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction. The amounts of TGF-β1 and VEGF released in the culture medium were assessed using enzyme-linked immunosorbent assay kits and normalized to the DNA content. Results: In response to 5 ng/ml FGF-2 for24 hours, the mRNA expression of VEGF increased at both culture phases (up to 6.1fold), whereas that of BMP-2 and TGF-β1 significantly increased only after the expansion (3.1-fold) or differentiation phase (2.1-fold), respectively. Similar trends were observed in the amounts of proteins measured in the culture medium. Conclusions: The authors’ results indicate that FGF-2 up-regulates the expression of BMP-2, TGF-β1, and VEGF in human bone marrow stromal cells, in a pattern dependent on the cell-differentiation stage. These findings prompt for in vivo investigations on the delivery of FGF-2 for the temporally/functionally regulated enhancement of bone marrow stromal cell–based bone induction.