Histopathological assessment of a two-stage reconstructive procedure of the infected Charcot foot.

INTRODUCTION: Charcot neuropathic osteoarthropathy (CN) can be complicated by osteomyelitis (OM). Surgery is a standard procedure to treat OM including debridement and interposition of antibiotic-loaded cement (ABLC) spacer. The course of CN and OM was investigated on a histopathological level. MATERIALS AND METHODS: Diabetic patients (n = 15) suffering from CN and midfoot OM underwent surgical debridement and interposition of ABLC was interposed. 6 weeks later, ABLC was removed and bone samples were taken again. Histopathological Charcot Score (HCS), Histopathological Osteomyelitis Evaluation Score (HOES) and microbiological assessment were used to evaluate osteomyelitic and neuroosteoarthropathic activity at both time points. RESULTS: Interposition of ABLC leads to microbiological/histopathological eradication of OM in 73%/87% of patients. CN activity-measured by HCS-could be reduced from moderate to low activity by ABLC spacer and correlated with HOES. CONCLUSIONS: CN activity could be reduced by surgery. It can be suggested that neuroosteoarthropathic activity measured by HCS is triggered by OM.

[Does inflammatory activity of Charcot foot affect successful corrective arthrodesis?] / Beeinflusst die Entzündungsaktivität des Charcot-Fußes eine erfolgreiche Korrekturarthrodese?

BACKGROUND: Neuro-osteoarthropathy of the foot (CN) is divided into an active and inactive stage, with impact on the further treatment. The histopathological Charcot score (HCS) can be used, to grade the inflammatory activity in tissue samples. RESEARCH QUESTION: This study aims to clarify whether successful bony healing after arthrodesis is related to inflammatory activity of the disease. MATERIAL AND METHOD: N = 80 patients underwent corrective arthrodesis of the midfoot (group 1) or hindfoot/ankle (group 2). A distinction was made between patients with/without diabetes mellitus and with/without pain perception. Intraoperative samples were taken to determine HCS. The osseous healing of the arthrodesis was determined by computed tomography 12 weeks postoperatively. RESULTS: There was an indirect correlation between bony consolidation and HCS. In group 2, there was a significantly worse bony healing in patients without pain sensation. There seems to be a tendency for HCS to be increased in patients without diabetes/no pain sensation. DISCUSSION: The present study confirms the assumption that corrective arthrodesis should be performed in the inactive stage of CN only. High activity levels obviously impede bony healing. HCS represents a relevant prognostic tool for surgical treatment.

A high-profile thread with grit-blasted and acid-etched surface reduces loosening of medial column fusion bolt in instable Charcot foot.

BACKGROUND: Intramedullary fusion bolts (FB) were introduced to stabilize the medial column of the instable Charcot foot (ICF), but complications as bolt loosening or breakage are frequent. We compared the use of a standard FB and a high-profile threaded FB with a grit-blasted surface. We hypothesized that implant related complications occurred less often and osseous consolidation of fusion sites was more distinctive using the latter type of FB. METHODS: Consecutive patients suffering from an ICF were stabilized with a high-profile threaded and surface-modified FB (HTFB) (n=20) or with a standard FB (n=20) which was placed into the first ray. Additional bolts and dorsal low-profile plates were applied in every patient. In a retrospective assessment osseous consolidation of the fusion sites was analyzed at 3 month and quantified by CT scan. At 3 and 12 month longitudinal foot arch collapse and rate of bolt loosening were assessed. RESULTS: Compared to the control group, the HTFB group reached significant higher consolidation after 3 month. No dislocation and a single bolt breakage was observed in the HTFB group after the fourth month, while the control group included 3 patients with bolt dislocation at 3±1 month and 5 patients with bolt breakage at 6±1.8 month. Compared to preoperative values, the improvement of Meary's angle after one-year was significant higher in the HTFB group (23.4°±14) than in controls (11.7°±13). CONCLUSIONS: Modification of bolt design improves the stability of the medial column: A higher rate of osseous consolidation of the medial column leads to lower rate of bolt dislocation/breakage and finally to permanently erected longitudinal foot arch. Initially disappointing results following medial column stabilization with fusion bolts can be rejected by modifications of bolt design and its technical application.

[Treatment of plantar, neuropathic and metatarsal ulcers by minimally invasive metatarsal osteotomy]. / Behandlung von plantaren, neuropathischen und metatarsalen Ulzera mittels distaler minimalinvasiver metatarsaler Osteotomie.

BACKGROUND: The chronic-mechanical plantar ulcer in diabetic-neuropathic foot syndrome is the starting point for severe infections of the foot and amputations. Frequent predilection sites are the metatarsal heads (MTH); in the context of neuropathy increased plantar peak pressure occurs and leads to an ulcer. GOAL: In this paper, we will examine whether minimally invasive pressure-reducing osteotomies, such as distal, minimally invasive metatarsal osteotomy (DMMO), can lead to the healing of metatarsal ulcers. Furthermore, the frequency of postoperative complications will be analyzed. METHOD: In a prospective study, n = 26 consecutive patients with plantar grade IA, IIA, and IIIA ulcers according to Wagner/Armstrong were included in the study under MTH 2, 3, 4 and 5 and with an unsuccessful conservative therapy >6 months. All patients received a DMMO of MT 2, 3, and 4, unless the ulcer was under MTH 5, then isolated DMMO MT 5 was performed. Clinical radiological check-ups took place over a follow-up interval of 26 ± 18 months (8-43 months). RESULTS: In all patients, the plantar ulcera healed after 5 ± 1 week, the recurrence rate was 8% and 3 patients had a transfer ulcer. Complications such as infection, pseudarthrosis or neuroosteoarthropathy did not occur. CONCLUSION: DMMO is an effective method for the treatment of recalcitrant ulcers under the metatarsal heads. The rate of a long-term cure is high; the complication rate is low; in ulcers under MTH 5, the DMMO should possibly be extended to the other metatarsal heads to reduce the risk of a transfer ulcer.

[Histopathological Charcot score on intraoperative tissue samples from the foot : A prospective investigation]. / Histopathologischer Charcot-Score an intraoperativen Gewebeproben vom Fuß : Prospektive Untersuchung.

BACKGROUND: Charcot neuropathy is a severe complication in patients with neuropathy. Without treatment, CN can lead to a destruction and collapse of the foot, with subsequent ulceration and infection. Finding an early diagnosis is essential and is based on clinical and radiological parameters (X-ray and MRI) because there is still no specific and reliable test. GOAL: Defining and validation of a Charcot score with defined histopathologic criteria. METHOD: Tissue samples from 37 surgeries (Charcot-group n = 20, control-group n = 17) from tarsal bones were taken prospectively. A semiquantitative histopathological score based on four defined criteria of fibrous-osteo-cartilage tissues (maximum 21 points) was defined, the scoring modalities were orientated towards the evaluated HOES score (histopathological osteomyelitis evaluation score) for osteomyelitis. A comparison of the Charcot-group with diabetes mellitus and verified CN as well as neuropathy with the control group with signs of CN or neuropathy was performed. RESULTS: Significant differences could be shown between the Charcot group and the control group in the score (10.5 vs 3.5 pts, p-value <0.001). There was a high significant correlation between the established tools for diagnostics of CN and the score (p-value <0,001). CONCLUSION: The histopathological Charcot score can detect a CN with high significance and correlates with high significance to established diagnostic tools for CN. It could represent a simple and cost-effective additive tool to verify CN in uncertain cases.

Gastrocnemius recession leads to medial shift of gait line, impairment of muscle strength and improved dorsal extension in forefoot overload syndrome.

BACKGROUND: Gastrocnemius recession (GR) has been introduced for treatment of forefoot overload syndrome (FOS). We questioned if GR leads to an altered forefoot load and weakness of plantarflexors in those patients. METHODS: 26 patients suffering from FOS and gastrocnemius tightness underwent GR. A strength power analysis of plantar flexors and a pedobarography was performed. Clinical outcome was measured by Foot Function Index (FFI). RESULTS: Plantarflexors are impaired about 40% six weeks and around 10% 24 weeks following GR compared to the contralateral side. Patients experienced a pain relief and an improvement of ankle dorsiflexion from 2° to 15°. An increased contact time of the heel (15%) and a shift of metatarsal plantar pressure from lateral to medial could be demonstrated. CONCLUSIONS: This study suggests that GR leads to pain reduction by an increase in heel contact time and a shift of gait line to medial in patients with a FOS. Despite, a temporary impairment of muscle strength has to be considered.

Gastrocnemius Recession Leads to Increased Ankle Motion and Improved Patient Satisfaction After 2 Years of Follow-Up.

The isolated gastrocnemius contracture present in neurologic healthy patients results in a significant limitation of ankle dorsiflexion causing pathologic gait patterns and a greater risk of further foot disorders. Gastrocnemius recession is an established procedure to increase ankle dorsiflexion. However, little evidence is available of the use of gastrocnemius recession in these patients. Complication rates, recurrence of gastrocnemius contracture, and the prevalence of additional foot disorders needs further evaluation. A study group of 64 operated limbs undergoing gastrocnemius recession was evaluated to determine the prevalence of foot disorders, pre- and postoperative ankle dorsiflexion, and incidence of complications. A subgroup of 15 (23.4%) patients without additional operative procedures was examined regarding ankle dorsiflexion, strength (Janda method), sensitivity in the operated limb, and the pre- and postoperative Foot Function Index scores. The prevalence of foot disorders showed pes planus (41%), hallux valgus (38%), metatarsalgia (19%), hammertoe deformity (13%), and symptomatic Haglund exostosis (11%). At 31 months of follow-up, the patients had significantly benefited from increased ankle dorsiflexion of 13.3° ± 7.9° (p < .001). Postoperatively, 16% patients experienced complications. In the subgroup of 15 patients, the follow-up examination after 44 months showed ankle dorsiflexion of 14° ± 7.1°. The plantarflexion strength was 4 of 5 (Janda method). The Foot Function Index score had improved significantly from 65.4 ± 26.5 points to 33.4 ± 19.5 points (p < .001). Patients with isolated gastrocnemius contracture seem to have a high prevalence of symptomatic foot disorders. At a mid-term follow-up examination, gastrocnemius recession (Strayer) was shown to be an effective procedure to significantly improve ankle dorsiflexion, functionality, and pain symptoms. More attention should be given to the development of postoperative complications.

Dynamic plantar pressure distribution, strength capacity and postural control after Lisfranc fracture-dislocation.

Substantial progress has been made in the operative treatment of Lisfranc fractures, however, the prognosis remains poor. We hypothesized that Lisfranc injuries change the postural control and muscle strength of the lower limb. Both are suggested to correlate with the clinical outcome and quality of life. 17 consecutive patients suffering from a Lisfranc fracture dislocation were registered, underwent open reduction and internal fixation and were followed-up for 50.5±25.7months (Mean±SDM). Biomechanical analysis of muscle strength capacities, postural control and plantar pressure distribution was assessed >6 month postoperatively. Results were correlated to clinical outcome (AOFAS, FFI, Pain, SF-36). The isokinetic assessment revealed a significant reduction in plantar flexor and dorsal extensor peak torque of the injured limb compared to the uninjured limb. The dorsal extensor peak torque thereby correlated well with clinical outcome. Altered postural control was evident by a significant reduction in unilateral stance time, from which we calculated a strong correlation between stance time and the isokinetic strength measurement. Plantar pressure measurements revealed a significant reduction in peak pressure under the midfoot and of Force-Time Integral beneath the second metatarsal. Sufficient rehabilitation is crucial to the clinical outcome following anatomical open reduction of Lisfranc fracture-dislocation. The present study supports a rehabilitation approach focussing on restoring proprioception and calf muscular strength including isometric exercises of the dorsal extensors.

Complication assessment and prevention strategies using midfoot fusion bolt for medial column stabilization in Charcot's osteoarthropathy.

In Charcot's osteoarthropathy stabilization of the medial column of the foot was introduced in order to establish a stable foot and reduce the risk for amputation. This study was performed to analyze postoperative complications, define risk factors for those and develop strategies for prevention. Since bolt dislocation takes place frequently, it was aimed to predict an appropriate time point for bolt removal under the condition that osseous healing has occurred. Fourteen consecutive patients with neuroosteoarthropathy of the foot and arch collapse were treated with open reduction and stabilization using midfoot fusion bolt and lateral lag screws. Age, gender, presence of preoperative osteomyelitis or ulcer, number of complications and operative revisions, Hba1c value, consolidation of arthrodesis, presence of a load-bearing foot and period to bolt dislocation was assessed. The mean follow-up was 21.4±14.6 (mean±SDM) months, 64% of patients suffered from diabetes with a preoperative Hba1c of 8.5±2.4. The mean number of revisions per foot was 3.6±4.1. Bolt dislocation was seen in 57% of the patients following 11.3±8.5 months; in 75% of these patients bony healing occurred before dislocation. There was a significant association between preoperative increased Hba1c value, presence of preoperative ulcer and wound infection. Healing of arthrodesis was demonstrated in 57% and a permanent weight-bearing foot without recurrent ulcer was achieved in 79%. The early and late postoperative complications could be controlled in general. A fully load-bearing and stable foot was obtained, despite osseous consolidation was not detected in all of these cases. Once a stable foot has established early removal of fusion bolt should be considered. To decrease the risk of infection Hba1c should be adjusted and ulcers should be treated before the operation.

Classification and Outcome of Fracture-Dislocation of the Cuneiform Bones.

Fractures and dislocations of the cuneiform bones are rare injuries to the midtarsal foot. The injury severity is often unclear, and the prognostic factors are unknown. The purpose of the present study was to characterize our insights of the diagnostics, therapy, and fracture patterns. We questioned whether the number of involved cuneiform bones and the type of injury would affect the clinical outcome. With this information, we aimed to develop a classification system for injuries of the cuneonavicular joint. Five patients who had sustained complex fracture-dislocation of the cuneiform bones were prospectively registered, underwent surgery, and were followed. We reviewed the published data and found 47 reports that included 55 patients to improve the informative value of our study. The injury mechanisms and therapy were evaluated, and the postoperative limitations and pain were assessed. The clinical outcome was correlated with the number of involved cuneiforms and the fracture/dislocation pattern. Direct trauma was associated with isolated fracture, and indirect injury was associated with isolated dislocations. Occasionally, these injuries were overlooked on conventional radiographs, and closed reduction frequently failed. The number of cuneiform bones involved and the type of injury were shown to affect the clinical outcome. We devised an easily applicable classification system for injuries to the cuneiform bones using this information. All cases were classified as isolated fractures (1), isolated dislocations (2), or fracture-dislocations (3) involving 1 (A), 2 (B), or 3 (C) cuneiform bones. The classification system we propose will facilitate a better understanding of the fracture patterns at the cuneonavicular joint line and is a good prognostic tool that requires validation in clinical settings.

Synovial cytokine expression in ankle osteoarthritis depends on age and stage.

PURPOSE: Aim of the study was the analysis of cytokine expression in ankle osteoarthritis (OA) hypothesizing age-dependent regulation patterns. METHODS: Forty-nine patients undergoing an arthroscopy of the ankle with different stages of chronic OA were prospectively included in a clinical trial comparing the group <18 years (n = 9, Ø15.1 ± 2.0 years) with the older patients (≥18 years, n = 40, Ø36.5 ± 11.9). Lavage fluids were analysed by ELISA for levels of aggrecan, BMP-2/7, IGF-1/R, bFGF, CD105, MMP-13, and IL-1ß. Additionally, clinical parameters and scores (FFI, CFSS, AOFAS) were evaluated and supplemented by radiographic scores [Kellgren-Lawrence Score (KLS) for conventional X-rays, Ankle Osteoarthritis Scoring System (AOSS) for MRI]. RESULTS: In contrast to distribution of gender and BMI (p < 0.005), parameters characterizing the cartilage defect as ICRS grading, size, and duration of symptoms were not dependent on age. The incidence of osteochondritis dissecans (OCD) was higher in the group <18 years (p < 0.006), but the average degree of OCD grading was not different. KLS and AOSS were significantly higher in the group ≥18 years (p < 0.02). Correlating with the higher degree of OA in the elderly, clinical function measured by FFI and AOFAS was statistically significantly worse (p < 0.05). Intra-articular concentrations of aggrecan (3.1-fold), bFGF (8.7-fold), BMP-7 (2.7-fold), and CD105 (1.5-fold) were statistically significantly higher in the group ≥18 years (p < 0.03). CONCLUSIONS: Confirming the hypothesis, increased synovial levels of aggrecan, bFGF, BMP-7, and CD105 were found in patients over 18 years. This correlated with a higher stage of OA determined by radiographic changes or deteriorated function and may offer starting points for new diagnostics and interventional strategies. LEVEL OF EVIDENCE: II.

Correlation of synovial cytokine expression with quality of cells used for autologous chondrocyte implantation in human knees.

The cell quality plays a decisive role in autologous chondrocyte implantation (ACI). Aim of the study was the analysis of in vivo interactions between synovial concentrations of cytokines and cell quality used for ACI. Knee lavage fluids of patients undergoing an ACI were examined for total protein content (TPC) and by ELISA for levels of basic fibroblast growth factor (bFGF), insulin-like growth factor 1, bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7). Cell quality following amplification for ACI was determined by surface expression of CD44, aggrecan, collagen type II and evaluation of cell characteristics. Data of 17 patients were supplemented by epidemiological parameters and clinical scores (IKDC, Lysholm, pain strength, subjective knee function). CD44 expression was positively associated with TPC and bFGF, and negatively linked to BMP-2 levels (p < 0.01). In contrast, expression of collagen type II did not show any statistically significant correlations with synovial protein concentrations. TPC was positively associated with intraarticular bFGF levels and pain strength (p < 0.01), both indicators for osteoarthritis (OA). Correlating with the negative relation of TPC and BMP-2, subjective knee function after 1 year was positively linked to intraarticular BMP-2 concentrations (p < 0.001). Similarly, expression of collagen type II indicated a favorable clinical result reaching statistical significance in case of pain strength (p < 0.01). Initially increased bFGF levels and CD44 expression indicated a worse clinical outcome after 1 year (IKDC, Lysholm Scores, pain strength). Surface expression of CD44 on chondrocytes used for ACI was negatively associated with synovial BMP-2 and positively to TPC and bFGF indicating catabolic synovial conditions. These correlations were also reflected by clinical outcome parameters.

Radiographic classification for fractures of the fifth metatarsal base.

OBJECTIVE: Avulsion fractures of the fifth metatarsal base (MTB5) are common fore foot injuries. Based on a radiomorphometric analysis reflecting the risk for a secondary displacement, a new classification was developed. MATERIALS AND METHODS: A cohort of 95 healthy, sportive, and young patients (age ≤ 50 years) with avulsion fractures of the MTB5 was included in the study and divided into groups with non-displaced, primary-displaced, and secondary-displaced fractures. Radiomorphometric data obtained using standard oblique and dorso-plantar views were analyzed in association with secondary displacement. Based on this, a classification was developed and checked for reproducibility. RESULTS: Fractures with a longer distance between the lateral edge of the styloid process and the lateral fracture step-off and fractures with a more medial joint entry of the fracture line at the MTB5 are at higher risk to displace secondarily. Based on these findings, all fractures were divided into three types: type I with a fracture entry in the lateral third; type II in the middle third; and type III in the medial third of the MTB5. Additionally, the three types were subdivided into an A-type with a fracture displacement <2 mm and a B-type with a fracture displacement ≥ 2 mm. A substantial level of interobserver agreement was found in the assignment of all 95 fractures to the six fracture types (κ = 0.72). The secondary displacement of fractures was confirmed by all examiners in 100%. CONCLUSIONS: Radiomorphometric data may identify fractures at risk for secondary displacement of the MTB5. Based on this, a reliable classification was developed.

Survival and neurologic outcome after traumatic out-of-hospital cardiopulmonary arrest in a pediatric and adult population: a systematic review.

INTRODUCTION: This systematic review is focused on the in-hospital mortality and neurological outcome of survivors after prehospital resuscitation following trauma. Data were analyzed for adults/pediatric patients and for blunt/penetrating trauma. METHODS: A systematic review was performed using the data available in Ovid Medline. 476 articles from 1/1964 - 5/2011 were identified by two independent investigators and 47 studies fulfilled the requirements (admission to hospital after prehospital resuscitation following trauma). Neurological outcome was evaluated using the Glasgow outcome scale. RESULTS: 34 studies/5391 patients with a potentially mixed population (no information was found in most studies if and how many children were included) and 13 paediatric studies/1243 children (age ≤ 18 years) were investigated. The overall mortality was 92.8% (mixed population: 238 survivors, lethality 96.7%; paediatric group: 237 survivors, lethality 86.4% = p < 0.001). CONCLUSIONS: Children have a higher chance of survival after resuscitation of an out-of-hospital traumatic cardiac arrest compared to adults but tend to have a poorer neurological outcome at discharge.

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions.

BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. RESULTS: The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). CONCLUSIONS: These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

Immunohistological localization of BMP-2, BMP-7, and their receptors in knee joints with focal cartilage lesions.

INTRODUCTION: Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. METHODS: Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. RESULTS: BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. CONCLUSIONS: BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

Nanomechanics of human adipose-derived stem cells: small GTPases impact chondrogenic differentiation.

OBJECTIVES: Human adipose-derived stem cells (ASCs) show gene expression of chondrogenic markers after three-dimensional cultivation. However, hypertrophy and osteogenic transdifferentiation are still limiting clinical applications. The aim of this study was to investigate the impact of small GTPases (Rac1 and RhoA) on transforming growth factor (TGF)-ß1-mediated chondrogenic differentiation of ASCs and compare it with BMP-2-induced hypertrophy, by assessing effects on intracellular and extracellular matrix. METHODS: In a novel experimental approach we characterized differentiation of living stem cells by single-cell elasticity measurements using atomic force microscopy. Results were matched with single-cell size measurements (diameter and volume) and quantitative real time-polymerase chain reaction for osteogenic and hypertrophic (alkaline phosphatase [ALP], collagen type X) as well as chondrogenic (collagen type II) gene expression. Intracellular F-actin expression was visualized by phalloidin staining of alginate-embedded ASCs. Statistical analysis was performed using analysis of variance (ANOVA) and two-sided t-test. RESULTS: Nontreated two-dimensional cultured ASCs (2D ASC) showed a significantly lower deformability than chondrocytes (Young's modulus: 294.4 vs. 225.1 Pa; ANOVA: p<0.001). Standard chondrogenic stimulation decreased stem cell elasticity to chondrocyte values (221.7 Pa). All other chondrogenic differentiated ASCs presented intermediate elasticity (BMP-2 stimulation: 269.1 Pa; Rac1 inhibition: 279.8 Pa; RhoA inhibtition: 257.8 Pa; p<0.05 compared to 2D ASC). F-actin fluorescence was visually decreased in Rac1-inhibited cells and increased in BMP-2-stimulated cells. Cell volume of 2D ASCs (6382.3 fL; p<0.001) was significantly higher than in all stimulated samples (BMP-2: 3076.7 fL; RhoA inhibition: 3126.0 fL). Volume of stem cells after standard chondrogenic stimulation (2590.0 fL) was not significantly different from chondrocyte volume (2244.9 fL). Rac1-Inhibitor reduced stem cell volume significantly below chondrocyte volume (1781.1 fL). Regarding mRNA expression, Rac1-Inhibitor reduced late hypertrophic transdifferentiation (collagen type X), while collagen type II production slightly increased (p<0.05). RhoA-Inhibitor increased osteogenesis (ALP) and slightly decreased collagen type II production (p<0.05). CONCLUSION: Biologically relevant nanomechanical parameters contribute to the evaluation of stem cell differentiation, in view of increased deformability of stem cells after chondrogenic stimulation. Regarding gene expression, Rac1 inhibition reduced hypertrophic chondrogenic differentiation and RhoA inhibition increased osteogenic transdifferentiation. Thus, the control of small GTPases is promising for cartilage tissue engineering purposes of stem cells.

Pain perception in knees with circumscribed cartilage lesions is associated with intra-articular IGF-1 expression.

BACKGROUND: Circumscribed cartilage defects are considered as prearthritic lesions and lead to differential intra-articular cytokine expression. Mechanisms of associated pain development and influence of smoking behavior are not yet fully understood in humans. PURPOSE: This study aimed to reveal relations between synovial cytokine levels in knees with circumscribed cartilage defects and pain sensation. STUDY DESIGN: Descriptive laboratory study. METHODS: In a clinical trial, knee lavage fluids of 42 patients with circumscribed cartilage lesions treated by either microfracturing (n = 19) or by autologous chondrocyte implantation (n = 23) and fluids of 5 healthy control individuals were prospectively collected. Preoperative knee pain was evaluated according to frequency and strength; subjective knee function was assessed using a visual analog scale and the International Knee Documentation Committee (IKDC) score. Synovial concentrations of aggrecan, insulin-like growth factor (IGF)-I, basic fibroblast growth factor (bFGF), interleukin (IL)-1ß, bone morphogenetic protein (BMP)-2, and BMP-7 were determined by enzyme-linked immunosorbent assay. RESULTS: Pain strength showed a highly significant association with intra-articular IGF-1 levels (ρ = .48, P < .01), but no correlation with synovial concentrations of aggrecan, bFGF, IL-1ß, BMP-2, and BMP-7. Although pain strength and frequency demonstrated a statistically significant relationship, no substantial association between pain frequency and any of the examined cytokine levels was found. Intra-articular IGF-1 concentrations significantly correlated with the area of cartilage damage (ρ = .35, P < .02); the other investigated cytokines failed to show this association. Neither of the determined intra-articular mediators demonstrated statistically significant correlations with subjective knee function or IKDC score. Only intra-articular concentrations of IGF-1 and BMP-2 statistically significantly correlated with age; total protein content was negatively associated with body mass index (P < .05). In smokers, synovial expression of total protein content, IGF-1, and bFGF was significantly diminished compared to nonsmokers (P < .05). CONCLUSION: Insulin-like growth factor-I is present in knees with circumscribed cartilage lesions in a size-dependent manner. IGF-1 levels correlated with indicators of pain perception; smoking negatively influenced synovial cytokine expression related to cartilage metabolism, but pain perception was not altered.

A new technique for seeding chondrocytes onto solvent-preserved human meniscus using the chemokinetic effect of recombinant human bone morphogenetic protein-2.

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering.

Association between expression of the bone morphogenetic proteins 2 and 7 in the repair of circumscribed cartilage lesions with clinical outcome.

BACKGROUND: Although there is much known about the role of BMPs in cartilage metabolism reliable data about the in vivo regulation in natural and surgically induced cartilage repair are still missing. METHODS: Lavage fluids of knee joints of 47 patients were collected during surgical therapy. 5 patients had no cartilage lesion and served as a control group, the other 42 patients with circumscribed cartilage defects were treated by microfracturing (19) or by an Autologous Chondrocyte Implantation (23). The concentrations of BMP-2 and BMP-7 were determined by ELISA. The clinical status was evaluated using the IKDC Score prior to and 1 year following the operation. RESULTS: High level expression in the control group was found for BMP-2, concentrations of BMP-7 remained below detection levels. No statistical differences could be detected in concentrations of BMP-2 or BMP-7 in the lavage fluids of knees with cartilage lesions compared to the control group. Levels of BMP-7 did not change after surgical cartilage repair, whereas concentrations of BMP-2 statistically significant increased after the intervention (p < 0.001). The clinical outcome following cartilage regenerating surgery increased after 1 year by 29% (p < 0.001). The difference of the IKDC score after 1 year and prior to the operation was used to quantify the degree of improvement following surgery. This difference statistically significant correlated with initial BMP-2 (R = 0.554, p < 0.001) but not BMP-7 (R = 0.031, n.s.) levels in the knee joints. CONCLUSIONS: BMP-2 seems to play an important role in surgically induced cartilage repair; synovial expression correlates with the clinical outcome.