Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Expression of antagonists of WNT and BMP signaling after non-rigid fixation of osteotomies

Delayed fracture healing and non-unions represent rare but severe complications in orthopedic surgery. Further knowledge on the mechanisms of the bone repair process and of the development of a pseudoarthrosis is essential to predict and prevent impaired healing of fractures. The present study aimed at elucidating differences in gene expression during the repair of rigidly and non-rigidly fixed osteotomies. For this purpose, the MouseFix™ and the FlexiPlate™ systems (AO Development Institute, Davos, CH), allowing the creation of well defined osteotomies in mouse femora, were employed. A time course following the healing process of the osteotomy was performed and bones and periimplant tissues were analyzed by high-resolution X-ray, MicroCT and by histology. For the assessment of gene expression, Low Density Arrays (LDA) were done. In animals with rigid fixation, X-ray and MicroCT revealed healing of the osteotomy within 3 weeks. Using the FlexiPlate™ system, the osteotomy was still visible by X-ray after 3 weeks and a stabilizing cartilaginous callus was formed. After 4.5 weeks, the callus was remodeled and the osteotomy was, on a histological level, healed. Gene expression studies revealed levels of transcripts encoding proteins associated with inflammatory processes not to be altered in tissues from bones with rigid and non-rigid fixation, respectively. Levels of transcripts encoding proteins of the extracellular matrix and essential for bone cell functions were not increased in the rigidly fixed group when compared to controls without osteotomy. In the FlexiPlate™ group, levels of transcripts encoding the same set of genes were significantly increased 3 weeks after surgery. Expression of transcripts encoding BMPs and BMP antagonists was increased after 3 weeks in repair tissues from bones fixed with FlexiPlate™, as were inhibitors of the WNT signaling pathways. Little changes only were detected in transcript levels of tissues from rigidly fixed bones. The data of the present study suggest that rigid fixation enables accelerated healing of an experimental osteotomy as compared to non-rigid fixation. The changes in the healing process after non-rigid fixation are accompanied by an increase in the levels of transcripts encoding inhibitors of osteogenic pathways and, probably as a consequence, by temporal changes in bone matrix synthesis.

Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Correlation of Pullout Force of Kirschner (K-) Wire to Other Factors Indicative of Bone Quality

More than 250,000 hip fractures occur annually in the United States and the most common fracture location is the femoral neck, the weakest region of the femur. Hip fixation surgery is conducted to repair hip fractures by using a Kirschner (K-) wire as a temporary guide for permanent bone screws. Variation has been observed in the force required to extract the K-wire from the femoral head during surgery. It is hypothesized that a relationship exists between the K-wire pullout force and the bone quality at the site of extraction. Currently, bone mineral density (BMD) is used as a predictor for bone quality and strength. However, BMD characterizes the entire skeletal system and does not account for localized bone quality and factors such as lifestyle, nutrition, and drug use. A patient’s BMD may not accurately describe the quality of bone at the site of fracture. This study aims to investigate a correlation between the force required to extract a K-wire from femoral head specimens and the quality of bone. A procedure to measure K-wire pullout force was developed and tested with pig femoral head specimens. The procedure was implemented on 8 human osteoarthritic femoral head specimens and the average pullout force for each ranged from 563.32 ± 240.38 N to 1041.01 ± 346.84 N. The data exhibited significant variation within and between each specimen and no statistically significant relationships were determined between pullout force and patient age, weight, height, BMI, inorganic to organic matter ratio, and BMD. A new testing fixture was designed and manufactured to merge the clinical and research environments by enabling the physician to extract the K-wire from each bone specimen himself. The new device allows the physician to gather tactile feedback on the relative ease of extraction while load history is recorded similar to the previous procedure for data acquisition. Future work will include testing human bones with the new device to further investigate correlations for predicting bone quality.

Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation

OBJECTIVES: One main problem occurring after bone grafting is resorption, leading to insufficient bone volume and quality, and may subsequently cause dental implant failure. Comparison of graft volume and bone density of iliac crest and calvarial transplants determined by animal studies demonstrates significantly lower resorption of bone grafts harvested from the skull. This paper is the first clinical study evaluating bone volume and density changes of calvarial split bone grafts after alveolar ridge reconstruction. MATERIAL AND METHODS: Bone volume and density were determined using CT scans and the software program Dicom Works in a total of 51 calvarial grafts after alveolar ridge augmentation in 15 patients. CT scans were taken in all 15 patients immediately after grafting (T0) and before implantation after a postoperative period of 6 months (T1). In five patients (26 calvarial grafts), a 1-year follow-up was performed (T2). RESULTS: A mean volume reduction of 16.2% at T1 (15 patients) and 19.2% at T2 (five patients) was observed. Bone density was high--about 1000 Hounsfield units--and did not change during the 1-year period. At the time of implantation, 41 transplants were classified as quality 1 bone and 10 as quality 2-3 bone. Grafting area and the technique used for grafting (inlay or onlay graft) did not affect the postoperative bone volume reduction. Generalized osteoporosis did not increase the resorption rate of calvarial transplants. CONCLUSION: Based on these findings, calvarial split bone grafts are a promising alternative for alveolar ridge reconstruction in dental implantology.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

The feasibility, efficacy and functional outcome of local anaesthetic repair of anterior and posterior vaginal wall prolapse

INTRODUCTION: Urogenital prolapse is a very common condition in women with a prevalence of 30%. If conservative therapy fails or is not desired by the patient, prolapse repair is usually performed under general or regional anaesthetic. The aim of the study was to evaluate feasibility, efficacy and functional outcome after fascial prolapse repairs under local anaesthetic (LA). PATIENTS AND METHODS: Between November 1999 and December 2000, 130 consecutive patients presenting with anterior or posterior prolapse or both were invited to have their procedure performed under LA. All patients with a symptomatic minimum stage II prolapse were included. Prior to surgery all women completed a standardized questionnaire examining the specific and non-specific symptoms of prolapse and their situation was classified using the ICS Pelvic Organ Prolapse (POP-Q) system. Follow up was 30 months. Objective success was defined as a stage 1 or less and no symptoms of bulge, subjective success was defined as lack of specific or non-specific symptoms of prolapse. RESULTS: There were 128 patients who agreed to have their operations performed under LA: 68 in the anterior group, 52 in the posterior group and 8 with a combined anterior and posterior repair. Objective cure rate was 88% for posterior repair, 87% for anterior repair and 63% for combined repair. Success rates were no different in primary from recurrent cases. There were no intraoperative complications and operating time was 21 min (anterior repair) or 23 min (posterior repair). There was no de novo postoperative urinary or stool incontinence and all patients but two would have the operation performed again under the same circumstances. The two remaining refused due to embarrassment but for no other reason. CONCLUSION: Local anaesthetic prolapse repair is feasible and effective in middle term results. It is well accepted by the patients who benefit from less side effects and short hospital stay.

Quality and quantity of bone following alveolar distraction osteogenesis in the human mandible

PURPOSE: The purpose of this prospective study on humans were to evaluate (a) the clinical outcome of alveolar distraction osteogenesis for the correction of vertically deficient edentulous mandibular ridges, (b) the clinical outcome of dental implants placed in the distracted areas, and (c) the quality and quantity of the bone that had formed in the distraction gap. MATERIAL AND METHODS: Seven patients presenting vertically deficient edentulous ridges were treated by means of distraction osteogenesis with an intraoral alveolar distractor. Approximately 3 months after consolidation of the distracted segments, 20 ITI solid screw SLA implants were placed in the distracted areas. Three to 4 months later, abutments were connected and prosthetic loading of the implants started. During implant site preparation, bone biopsies were taken at the implant sites with trephine burrs for histologic and histometric analyses. RESULTS: The mean follow-up after the initial prosthetic loading was 18 months (range 12-24 months). The mean bone gain obtained at the end of distraction was 7 mm (range 5-9 mm). The cumulative success rate of implants 2 years after the onset of prosthetic loading was 95%, whereas the survival rate of implants was 100%. The newly formed bone consisted of woven bone reinforced by parallel-fibered bone with bone marrow spaces between the bone trabeculae. The bone area fraction in the distraction region ranged from 21.6% to 57.8% (38.5+/-11.7%). DISCUSSION AND CONCLUSIONS: Results from this study showed that (a) distraction osteogenesis is a reliable technique for the correction of vertically deficient edentulous ridges, (b) the regenerated bone withstood the functional demands of implant loading, (c) survival and success rates of implants placed in the distracted areas were consistent with those of implants placed in native bone, and (d) there is sufficient bone volume and maturity in the distracted region for primary stability of the implant.

MRI characteristics and histology of bone marrow lesions in dogs with experimentally induced osteoarthritis

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Post-operatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.

Evaluation of reparative cartilage after autologous chondrocyte implantation for osteochondritis dissecans: histology, biochemistry, and MR imaging

BACKGROUND: The aim of this study was to investigate the biochemical properties, histological and immunohistochemical appearance, and magnetic resonance (MR) imaging findings of reparative cartilage after autologous chondrocyte implantation (ACI) for osteochondritis dissecans (OCD). METHODS: Six patients (mean age 20.2 +/- 8.8 years; 13-35 years) who underwent ACI for full-thickness cartilage defects of the femoral condyle were studied. One year after the procedure, a second-look arthroscopic operation was performed with biopsy of reparative tissue. The International Cartilage Repair Society (ICRS) visual histological assessment scale was used for histological assessment. Biopsied tissue was immunohistochemically analyzed with the use of monoclonal antihuman collagen type I and monoclonal antihuman collagen type II primary antibodies. Glycosaminoglycan (GAG) concentrations in biopsied reparative cartilage samples were measured by high performance liquid chromatography (HPLC). MR imaging was performed with T1- and T2-weighted imaging and three-dimensional spoiled gradient-recalled (3D-SPGR) MR imaging. RESULTS: Four tissue samples were graded as having a mixed morphology of hyaline and fibrocartilage while the other two were graded as fibrocartilage. Average ICRS scores for each criterion were (I) 1.0 +/- 1.5; (II) 1.7 +/- 0.5; (III) 0.6 +/- 1.0; (IV) 3.0 +/- 0.0; (V) 1.8 +/- 1.5; and (VI) 2.5 +/- 1.2. Average total score was 10.7 +/- 2.8. On immunohistochemical analysis, the matrix from deep and middle layers of reparative cartilage stained positive for type II collagen; however, the surface layer did not stain well. The average GAG concentration in reparative cartilage was 76.6 +/- 4.2 microg/mg whereas that in normal cartilage was 108 +/- 11.2 microg/mg. Common complications observed on 3D-SPGR MR imaging were hypertrophy of grafted periosteum, edema-like signal in bone marrow, and incomplete repair of subchondral bone at the surgical site. Clinically, patients had significant improvements in Lysholm scores. CONCLUSIONS: In spite of a good clinical course, reparative cartilage after ACI had less GAG concentration and was inferior to healthy hyaline cartilage in histological and immunohistochemical appearance and on MRI findings.

Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: A novel biphasic calcium phosphate (CaP) granulate consisting of hydroxyapatite (HA) and beta-tricalciumphosphate (TCP) was compared with pure HA and pure TCP and with autograft as positive control. MATERIALS AND METHODS: Four standardized bone defects were prepared in both mandibular angles of 16 minipigs and grafted with autogenous bone chips, HA, HA/TCP (60% : 40%), or TCP. Histologic and histomorphometric analysis of bone formation and graft degradation followed healing periods of 2, 4, 8, and 24 weeks. RESULTS: 2 weeks: more bone formation in defects filled with autograft than with the three CaP materials (P<0.05). 4 weeks: bone formation differed significantly (P<0.05) between all four materials (autograft>TCP>HA/TCP>HA). 8 weeks: more bone formation in defects with autograft and TCP than with HA/TCP (P<0.05), and HA/TCP had more bone formation than HA (P<0.05). 24 weeks: no difference in bone formation between the groups. Autograft and TCP resorbed quickly and almost completely over 8 weeks, whereas HA/TCP and HA showed limited degradation over 24 weeks. CONCLUSION: All defects healed with mature lamellar bone and intimate contact between bone and the remaining graft material. The rate of bone formation corresponded to the content of TCP in the CaP materials.

Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures

Early implant placement is one treatment option for implant therapy following single-tooth extraction in the anterior maxilla. The surgical technique presented here is characterized by tooth extraction without flap elevation, a 4- to 8-week soft tissue healing period, implant placement in a correct three-dimensional position, simultaneous contour augmentation on the facial aspect with guided bone regeneration using a bioabsorbable collagen membrane combined with autogenous bone chips and a low-substitution bone filler, and tension-free primary wound closure. The surgical step-by-step procedure is presented with a case report. In addition, the biologic rationale is discussed.

The influence of non-matching implant and abutment diameters on radiographic crestal bone levels in dogs

BACKGROUND: It has been shown that different implant designs and different vertical implant positions have an influence on crestal bone levels. The aim of the present study was to evaluate radiographic crestal bone changes around experimental dental implants with non-matching implant-abutment diameters placed submucosally or transmucosally at three different levels relative to the alveolar crest. METHODS: Sixty two-piece dental implants with non-matching implant-abutment diameters were placed in edentulous spaces bilaterally in five foxhounds. The implants were placed submucosally or transmucosally in the left or the right side of the mandible. Within each side, six implants were randomly placed at three distinct levels relative to the alveolar crest. After 12 weeks, 60 crowns were cemented. Radiographs were obtained from all implant sites following implant placement, after crown insertion, and monthly for 6 months after loading. RESULTS: Radiographic analysis revealed very little bone loss and a slight increase in bone level for implants placed at the level of the crest or 1 mm above. The greatest bone loss occurred at implants placed 1 mm below the bone crest. No clinically significant differences regarding marginal bone loss and the level of the bone-to-implant contact were detected between implants with a submucosal or a transmucosal healing. CONCLUSIONS: Implants with non-matching implant-abutment diameters demonstrated some bone loss; however, it was a small amount. There was no clinically significant difference between submucosal and transmucosal approaches.