Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism.

OBJECTIVE: Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. DESIGN: Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. RESULTS: OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. CONCLUSION: Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

Histological and molecular features of the subacromial bursa of rotator cuff tears compared to non-tendon defects: a pilot study.

BACKGROUND: The role of the subacromial bursa in the development or healing of shoulder pathologies is unclear. Due to this limited knowledge, we aimed to understand specific reactions of the subacromial bursa according to rotator cuff (RC) pathologies compared to non-tendon defects of the shoulder. We hypothesized that the tissue composition and inflammatory status of the bursa are likely to vary between shoulder pathologies depending on the presence and the extent of RC lesion. METHOD: Bursa samples from patients with either 1) shoulder instability with intact RC (healthy bursa, control), 2) osteochondral pathology with intact RC, 3) partial supraspinatus (SSP) tendon tear, or 4) full-thickness SSP tear were investigated histologically and on gene expression level. RESULT: Bursae from SSP tears differed from non-tendon pathologies by exhibiting increased chondral metaplasia and TGFß1 expression. MMP1 was not expressed in healthy bursa controls, but strongly increased with full-thickness SSP tears. Additionally, the expression of the inflammatory mediators IL1ß, IL6, and COX2 increased with the extent of SSP tear as shown by correlation analysis. In contrast, increased angiogenesis and nerve fibers as well as significantly upregulated IL6 and COX2 expression were features of bursae from patients with osteochondral pathology. Using immunohistochemistry, CD45+ leukocytes were observed in all examined groups, which were identified in particular as CD68+ monocytes/macrophages. CONCLUSION: In summary, besides the strong increase in MMP1 expression with SSP tear, molecular changes were minor between the investigated groups. However, expression of pro-inflammatory cytokines correlated with the severity of the SSP tear. Most pronounced tissue alterations occurred for the osteochondral pathology and full-thickness SSP tear group, which demonstrates that the bursal reaction is not exclusively dependent on the occurrence of an SSP tear rather than longstanding degenerative changes. The present bursa characterization contributes to the understanding of specific tissue alterations related to RC tears or non-tendon shoulder pathologies. This pilot study provides the basis for future studies elucidating the role of the subacromial bursa in the development or healing of shoulder pathologies.

The Effect of Age and Intrinsic Aerobic Exercise Capacity on the Expression of Inflammation and Remodeling Markers in Rat Achilles Tendons.

Old age, adiposity, and metabolic disorders are known as risk factors for chronic tendinopathy, which is a common problem in both athletes and the general population. However, the importance of these influencing factors has not yet been well understood. This study investigated alterations in gene expression and histology of Achilles tendons of young (10 weeks) and old (100 weeks) rats bred for low (low capacity runners, LCR) and high (high capacity runners, HCR) intrinsic aerobic exercise capacity. In this rat model, LCR displayed a phenotype of reduced exercise capacity, higher body weight, and metabolic dysfunctions compared to HCR. We hypothesized that the risk factors for tendinopathy in old LCR could lead to more pronounced impairments in Achilles tendon tissue. In quantitative real-time PCR (qPCR), age-related downregulation of tenocyte markers e.g., tenomodulin, genes related to matrix modeling and remodeling (e.g., collagens, elastin, biglycan, fibronectin, tenascin C) as well as transforming growth factor beta 3 (Tgfb3) have been detected. Inflammation marker cyclooxygenase 2 (Cox2) was downregulated in old rats, while microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in old HCR and old LCR. In all groups, interleukin 6 (Il6), interleukin 1 beta (Il1b), and tumor necrosis factor alpha (Tnfa) showed no significant alteration. In histological evaluation, tendons of old rats had fewer and more elongated tenocyte nuclei than young rats. Even though a higher content of glycosaminoglycans, a sign of degeneration, was found in old HCR and LCR, no further signs of tendinopathy were detectable in tendons of old rats by histological evaluation. Low intrinsic aerobic exercise capacity and the associated phenotype did not show significant effects on gene expression and tendon histology. These findings indicate that aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue and suggests that other risk factors associated with intrinsic aerobic exercise capacity are less influential in this rat model.

A new sequential animal model for infection-related non-unions with segmental bone defect.

BACKGROUND: The treatment of fracture-related infections (FRI) is still a challenge for orthopedic surgeons. The prevalence of FRI is particularly high in open fractures with extensive soft-tissue damage. This study aimed to develop a new two-step animal model for non-unions with segmental bone defects, which could be used to evaluate new innovative bone substitutes to improve the therapeutic options in humans with FRI and bone defects. METHODS: After randomization to infected or non-infected groups, 30 Sprague-Dawley rats underwent a transverse osteotomy of the mid-shaft femur with a 5 mm defect. Additionally, the periosteum at the fracture zone was cauterized at both sides. After intramedullary inoculation with 103 CFU Staphylococcus aureus (infected group) or PBS (non-infected group), a fracture stabilization was done by intramedullary K-wires. After 5 weeks, the bone healing process was evaluated, and revision surgery was performed in order to obtain increased bone healing. The initial K-wires were removed, and debridement of the osteotomy-gap was done followed by a more stable re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and the bone consolidation was tested biomechanically and the callus formation quantitatively by micro-CT analysis. RESULTS: We developed and presented a new two-stage non-union animal model through a targeted S. aureus infection. After 5 weeks, all animals showed a non-union irrespective of assignment to the infected and non-infected group. Lane and Sandhu score showed a higher callus formation in the infected group. In all infected animals, the inoculated S. aureus strain was detected in the revision surgery. The second surgery did not improve bone healing, as shown by the Lane Sandhu score and in the µ-CT analysis. Similarly, biomechanical testing showed in both groups a significantly lower maximum torque as compared to the contralateral side (p < 0.0001). CONCLUSIONS: We were able to successfully develop a new two-stage non-union animal model, which reflects a genuine clinical situation of an infection-related non-union model with segmental bone defects. This model could be used to evaluate various therapeutic anti-infectious and osteoinductive strategies in FRIs.

Editorial of Special Issue: Biological Basis of Musculoskeletal Regeneration 2019.

The Special Issue "Biological Basis of Musculoskeletal Regeneration 2019" aimed to collect research and review articles that cover various aspects of the molecular and cellular mechanisms of bone, cartilage, tendon/ligament, and muscle regeneration [...].

In Vivo and In Vitro Mechanical Loading of Mouse Achilles Tendons and Tenocytes-A Pilot Study.

Mechanical force is a key factor for the maintenance, adaptation, and function of tendons. Investigating the impact of mechanical loading in tenocytes and tendons might provide important information on in vivo tendon mechanobiology. Therefore, the study aimed at understanding if an in vitro loading set up of tenocytes leads to similar regulations of cell shape and gene expression, as loading of the Achilles tendon in an in vivo mouse model. In vivo: The left tibiae of mice (n = 12) were subject to axial cyclic compressive loading for 3 weeks, and the Achilles tendons were harvested. The right tibiae served as the internal non-loaded control. In vitro: tenocytes were isolated from mice Achilles tendons and were loaded for 4 h or 5 days (n = 6 per group) based on the in vivo protocol. Histology showed significant differences in the cell shape between in vivo and in vitro loading. On the molecular level, quantitative real-time PCR revealed significant differences in the gene expression of collagen type I and III and of the matrix metalloproteinases (MMP). Tendon-associated markers showed a similar expression profile. This study showed that the gene expression of tendon markers was similar, whereas significant changes in the expression of extracellular matrix (ECM) related genes were detected between in vivo and in vitro loading. This first pilot study is important for understanding to which extent in vitro stimulation set-ups of tenocytes can mimic in vivo characteristics.

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFß) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFß signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

The effect of autologous platelet rich plasma on tenocytes of the human rotator cuff.

BACKGROUND: Platelet rich plasma (PRP) is widely used in rotator cuff repairs but its effect on the healing process is unclear. Several cell culture studies on the effect of allogenic PRP have reported promising results but are not transferable to clinical practice. The aim of the present study is to assess the possible effect of autologous PRP on rotator cuff tendon cells. The amount of growth factors involved with tendon-bone healing (PDGF-AB, IGF-1, TGF-ß1, BMP-7 and -12) is quantified. METHODS: Rotator cuff tissue samples were obtained from (n = 24) patients grouped by age (>/< 65 years) and sex into four groups and cells were isolated and characterized. Later, autologous PRP preparations were obtained and the effect was analyzed by means of cell proliferation, collagen I synthesis and expression of collagen I and III. Furthermore, the PRPs were quantified for growth factor content by means of platelet-derived growth factor (PDGF-AB), insulin-like growth factor (IGF-1), transforming growth factor (TGF-ß1), as well as bone morphogenetic protein (BMP) -7 and - 12. RESULTS: Cell proliferation and absolute synthesis of collagen I were positively affected by PRP exposure compared to controls (p < 0.05), but expression and relative synthesis of collagen I (normalized to cell proliferation) were significantly reduced. PRP contained high amounts of IGF-1 and lower levels of TGF-ß1 and PDGF-AB. The amounts of BMP-7 and -12 were below the detection limits. CONCLUSIONS: PRP is a source of growth factors such involved with tendon-bone healing. PRP had an anabolic effect on the human rotator cuff tenocytes of the same individual in vitro by means of cell proliferation and absolute, but not relative collagen I synthesis. These results encourage further studies on clinical outcomes with more comparable standards in terms of preparation and application methods. LEVEL OF EVIDENCE: Controlled laboratory study.

Bone morphogenetic proteins - 7 and - 2 in the treatment of delayed osseous union secondary to bacterial osteitis in a rat model.

BACKGROUND: Bone infections due to trauma and subsequent delayed or impaired fracture healing represent a great challenge in orthopedics and trauma surgery. The prevalence of such bacterial infection-related types of delayed non-union is high in complex fractures, particularly in open fractures with additional extensive soft-tissue damage. The aim of this study was to establish a rat model of delayed osseous union secondary to bacterial osteitis and investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in the situation of an ongoing infection. METHODS: After randomization to four groups 72 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia stabilized by intramedullary titanium K-wires. Three groups received an intramedullary inoculation with Staphylococcus aureus (103 colony-forming units) before stabilization and the group without bacteria inoculation served as healing control. After 5 weeks, a second surgery was performed with irrigation of the medullary canal and local rhBMP-7 and rhBMP-2 treatment whereas control group and infected control group received sterile saline. After further 5 weeks rats were sacrificed and underwent biomechanical testing to assess the mechanical stability of the fractured bone. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, and to quantify callus formation and the mineralized area of the callus. RESULTS: Biomechanical testing showed a significantly higher fracture torque in the non-infected control group and the infected rhBMP-7- and rhBMP-2 group compared with the infected control group (p < 0.001). RhBMP-7 and rhBMP-2 groups did not show statistically significant differences (p = 0.57). Histological findings supported improved bone-healing after rhBMP treatment but quantitative micro-CT and histomorphometric results still showed significantly more hypertrophic callus tissue in all three infected groups compared to the non-infected group. Results from a semiquantitative bone-healing-score revealed best bone-healing in the non-infected control group. The expected chronic infection was confirmed in all infected groups. CONCLUSIONS: In delayed bone healing secondary to infection rhBMP treatment promotes bone healing with no significant differences in the healing efficacy of rhBMP-2 and rhBMP-7 being noted. Further new therapeutic bone substitutes should be analyzed with the present rat model for delayed osseous union secondary to bacterial osteitis.

Different Achilles Tendon Pathologies Show Distinct Histological and Molecular Characteristics.

Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7), chronic ruptures (n = 6), acute ruptures (n = 13), and intact tendons (n = 4) were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR) analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases) in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL) 1ß, tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor ß1, cyclooxygenase 2), inflammatory cells (cluster of differentaition (CD) 3, CD68, CD80, CD206), fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin), and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor) were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies.

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study.

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1ß, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture.

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2-4 days), middle (5-6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process.

Testing of antibiotic releasing implant coatings to fight bacteria in combat-associated osteomyelitis - an in-vitro study.

PURPOSE: Surgical procedures to prevent osteomyelitis after trauma can be supported by local application of antibiotics. This in-vitro study investigated the release and impact of antibiotics from implant coatings against bacteria associated with combat-related osteomyelitis. METHODS: K-wires were coated with poly(D,L-lactide) and ciprofloxacin, gentamicin, colistin, daptomycin or cefoxitin in different concentrations. The release was quantified and antimicrobial activity tested for different gram-positive or gram-negative bacteria, alone and in combination. To exclude toxic effects, primary osteoblast-like cells were exposed to antibiotic coating concentrations. RESULTS: All antibiotics alone and in combination showed an initial burst release with dose dependent antimicrobial activity and no negative effects on osteoblast-like cells, except for cefoxitin. CONCLUSIONS: Implant coatings can be customized with single or double antibiotic coatings to effectively fight different bacteria and also mixed infections in the treatment of a combat-acquired osteomyelitis. However, optimal drug load and degradation behaviour of individual antibiotics have to be considered.

Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

An imbalance between matrix metalloproteases (MMPs) and the tissue inhibitors of metalloproteases (TIMPs) may have a negative impact on the healing of rotator cuff tears. The aim of the project was to assess a possible relationship between clinical and radiographic characteristics of patients such as the age, sex, as well as the degenerative status of the tendon and the MMPs and TIMPs in their tenocyte-like cells (TLCs). TLCs were isolated from ruptured supraspinatus tendons and quantitative Real-Time PCR and ELISA was performed to analyze the expression and secretion of MMPs and TIMPs. In the present study, MMPs, mostly gelatinases and collagenases such as MMP-2, -9 and -13 showed an increased expression and protein secretion in TLCs of donors with higher age or degenerative status of the tendon. Furthermore, the expression and secretion of TIMP-1, -2 and -3 was enhanced with age, muscle fatty infiltration and tear size. The interaction between MMPs and TIMPs is a complex process, since TIMPs are not only inhibitors, but also activators of MMPs. This study shows that MMPs and TIMPs might play an important role in degenerative tendon pathologies.

Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver-bone axis.

Patients with chronic liver diseases frequently exhibit decreased bone mineral densities (BMD), which is defined as hepatic osteodystrophy (HOD). HOD is a multifactorial disease whose regulatory mechanisms are barely understood. Thus, an early diagnosis and therapy is hardly possible. Therefore, the aim of our study consisted in characterizing a mouse model reflecting the human pathomechanism. Serum samples were collected from patients with chronic liver diseases and 12-week old C57Bl6/N mice after 6-week treatment with carbon tetrachloride (CCl4). Repetitive injections of CCl4 induced liver damage in mice, resembling liver fibrosis in patients, as assessed by serum analysis and histological staining. Although CCl4 did not affect primary osteoblast cultures, µCT analysis revealed significantly decreased BMD, bone volume, trabecular number and thickness in CCl4-treated mice. In both HOD patients and CCl4-treated mice, an altered vitamin D metabolism with decreased CYP27A1, CYP2R1, vitamin D-binding protein GC and increased 7-dehydrocholesterol reductase hepatic gene expression, results in decreased 25-OH vitamin D serum levels. Moreover, both groups exhibit excessively high active transforming growth factor-beta (TGF-ß) serum levels, inhibiting osteoblast function in vitro. Summarizing, our mouse model presents possible mediators of HOD, e.g. altered vitamin D metabolism and increased active TGF-ß. Liver damage and significant changes in bone structure and mineralization are already visible by µCT analysis after 6 weeks of CCl4 treatment. This fast response and easy transferability makes it an ideal model to investigate specific gene functions in HOD.

Efficacy of two different demineralised bone matrix grafts to promote bone healing in a critical-size-defect: a radiological, histological and histomorphometric study in rat femurs.

PURPOSE: The aim of the study was to compare two different demineralised bone matrices used clinically regarding their ability to induce bone healing in a critical-size-defect rat model. METHODS: We stabilised 4 mm femur defects with a custom-made plate and filled them either with demineralised bone matrix (DBM) or DBX (DBX Putty®). Bone morphogenetic protein 2 (BMP-2)-loaded collagen and an empty defect served as controls. The outcome was followed after 21 and 42 days by radiology (Faxitron; microCT) and histology. RESULTS: Defect healing did not occur in any animal from the empty control, DBM or DBX group. Residuals of the implanted material were still found after six weeks, but only limited callus formation was visible. In contrast, the BMP-2 control demonstrated enhanced formation of callus tissue and undisturbed healing. After 21 days, 11 out of 16 and after 42 days, 7 out of 8 BMP-2-treated animals showed complete defect bridging by cancellous bone tissue. CONCLUSIONS: Demineralised bone grafts were not capable of defect reconstruction; only BMP-2 was able to provide sufficient stimulus to induce uneventful bridging under the specific experimental conditions.

A pilot study investigating the histology and growth factor content of human non-union tissue.

PURPOSE: The reason for the formation of an atrophic non-union is not clear and an altered vascularization as well as a deregulation of endogenous growth factors is hypothesized. To obtain more information, we analysed human non-union tissue regarding the histology and quantity of several growth factors. METHODS: Tissue from patients with an atrophic non-union (n = 44) or with a healed fracture (n = 13) was analysed. Using histological and immunohistochemical methods the tissue composition was investigated. On the protein level the amount of several growth factors important for bone healing was analysed. RESULTS: The tissue composition was very inhomogeneous containing fibrous, cartilaginous and bony tissue. Vessels were present in all investigated samples without a difference between the tissue from non-union and control patients. The growth factor BMP-2 was below the detection limit in all samples, whereas IL-6 and IGF-I were measured only in a few samples of both groups. TGF-ß1, VEGF-A and BMP-4 were detectable in the majority of the samples of both groups with a high variability in the amount but no difference between the groups. The quantity of both growth factors, BMP-7 and PDGF-AB, was significantly lower in the non-union tissue compared to the healed controls. CONCLUSION: The reduced quantity of BMP-7 and PDGF-AB might be responsible for the impaired healing. Further studies analysing material from more patients and investigating the early healing phases, however, are necessary to obtain further information and consequently improve healing strategies.

Stimulation of bone healing by sustained bone morphogenetic protein 2 (BMP-2) delivery.

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (µCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p=0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p=0.006) with reduced cartilage at Day 84 (p=0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p=0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.