Hans Polzer

Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), β(1) and β(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.

Diagnosis and treatment of acute ankle injuries: development of an evidence-based algorithm

Acute ankle injuries are among the most common injuries in emergency departments. However, there are still no standardized examination procedures or evidence-based treatment. Therefore, the aim of this study was to systematically search the current literature, classify the evidence, and develop an algorithm for the diagnosis and treatment of acute ankle injuries. We systematically searched PubMed and the Cochrane Database for randomized controlled trials, meta-analyses, systematic reviews or, if applicable, observational studies and classified them according to their level of evidence. According to the currently available literature, the following recommendations have been formulated: i) the Ottawa Ankle/Foot Rule should be applied in order to rule out fractures; ii) physical examination is sufficient for diagnosing injuries to the lateral ligament complex; iii) classification into stable and unstable injuries is applicable and of clinical importance; iv) the squeeze-, crossed leg- and external rotation test are indicative for injuries of the syndesmosis; v) magnetic resonance imaging is recommended to verify injuries of the syndesmosis; vi) stable ankle sprains have a good prognosis while for unstable ankle sprains, conservative treatment is at least as effective as operative treatment without the related possible complications; vii) early functional treatment leads to the fastest recovery and the least rate of reinjury; viii) supervised rehabilitation reduces residual symptoms and re-injuries. Taken these recommendations into account, we present an applicable and evidence-based, step by step, decision pathway for the diagnosis and treatment of acute ankle injuries, which can be implemented in any emergency department or doctors practice. It provides quality assurance for the patient and promotes confidence in the attending physician.

Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation.

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing. Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining. Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation. Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients.

Accelerated rehabilitation following Achilles tendon repair after acute rupture - Development of an evidence-based treatment protocol

The acute rupture of the Achilles tendon is a protracted injury. Surgery is only the beginning of a long rehabilitation period. Therefore, the rehabilitation protocol is an integral aspect to restore the pre-injury activity level. Despite several trials available comparing different treatment regimes, there is still no consensus regarding the optimal protocol. Consequently, the aim of our study was to systematically search the evidence available and define a precise rehabilitation programme after operative repair of acute Achilles tendon rupture based on the trials with the highest level of evidence. We performed a systematic literature search in Medline, Embase and Cochrane library. We identified twelve randomized controlled trials comparing different treatment regimes after operative repair of the Achilles tendon. Five trials compared full to non weight bearing, all applying immobilization in equinus. Immediate full weight bearing led to significant higher patient satisfaction, earlier ambulation and return to pre-injury activity. Four trials compared early ankle mobilization to immobilization. All trials found mobilization to be superior as it shortens time to return to work and sports significantly. Three trials compared the combination of full weight bearing and early ankle mobilization to immobilization. This combination was most beneficial. Patients showed significantly higher satisfaction, less use of rehabilitation resources, earlier return to pre-injury activities and further demonstrated significantly increased calf muscle strength, reduced atrophy and tendon elongation. No study found an increased rerupture rate for the more progressive treatment. In conclusion, the rehabilitation protocol after Achilles tendon repair should allow immediate full weight bearing. After the second postoperative week controlled ankle mobilization by free plantar flexion and limited dorsiflexion at 0° should be applied.

Overcoming hypoxia in 3D culture systems for tissue engineering of bone in vitro using an automated, oxygen-triggered feedback loop

Tissue engineering is an attractive approach to heal bony defects. However, three-dimensional cell-scaffold constructs display uneven oxygen supply resulting in inhomogeneous tissue quality. We assessed different strategies to improve oxygen supply in vitro. Scaffolds with differing inner surface were seeded with preosteoblastic cells and cultivated either statically or in perfusion bioreactors. Oxygen concentration and pH were measured in the center of the scaffolds. An inductive feedback mechanism was build to increase bioreactor pump speed according to the oxygen concentrations measured within the scaffolds. While pH remained stable, oxygen concentration decreased significantly under static conditions within the cell-seeded scaffolds. Reducing the scaffolds’ inner surface as well as increasing perfusion speeds in bioreactors resulted in improved oxygen supply. We conclude that improving oxygen supply to three dimensional culture systems for bone tissue engineering is feasible in an automated manner. Culture conditions have to be adapted to each cell-scaffold system individually.

Hallux rigidus: Joint preserving alternatives to arthrodesis - a review of the literature.

Hallux rigidus describes the osteoarthritis of the first metatarsophalangeal joint. It was first mentioned in 1887. Since then a multitude of terms have been introduced referring to the same disease. The main complaints are pain especially during movement and a limited range of motion. Radiographically the typical signs of osteoarthritis can be observed starting at the dorsal portion of the joint. Numerous classifications make the comparison of the different studies difficult. If non-operative treatment fails to resolve the symptoms operative treatment is indicated. The most studied procedure with reproducible results is the arthrodesis. Nevertheless, many patients refuse this treatment option, favouring a procedure preserving motion. Different motion preserving and joint sacrificing operations such as arthroplasty are available. In this review we focus on motion and joint preserving procedures. Numerous joint preserving osteotomies have been described. Most of them try to relocate the viable plantar cartilage more dorsally, to decompress the joint and to increase dorsiflexion of the first metatarsal bone. Multiple studies are available investigating these procedures. Most of them suffer from low quality, short follow up and small patient numbers. Consequently the grade of recommendation is low. Nonetheless, joint preserving procedures are appealing because if they fail to relief the symptoms an arthrodesis or arthroplasty can still be performed thereafter.

The influence of knee position on ankle dorsiflexion - a biometric study

BackgroundMusculus gastrocnemius tightness (MGT) can be diagnosed by comparing ankle dorsiflexion (ADF) with the knee extended and flexed. Although various measurement techniques exist, the degree of knee flexion needed to eliminate the effect of the gastrocnemius on ADF is still unknown. The aim of this study was to identify the minimal degree of knee flexion required to eliminate the restricting effect of the musculus gastrocnemius on ADF.MethodsBilateral ADF of 20 asymptomatic volunteers aged 18-40 years (50% female) was assessed prospectively at six different degrees of knee flexion (0°, 20°, 30°, 45°, 60°, 75°, Lunge). Tests were performed following a standardized protocol, non weightbearing and weightbearing, by two observers. Statistics comprised of descriptive statistics, t-tests, repeated measurement ANOVA and ICC.Results20 individuals with a mean age of 27 ± 4 years were tested. No significant side to side differences were observed. The average ADF [95% confidence interval] for non weightbearing was 4° [1°-8°] with the knee extended and 20° [16°-24°] for the knee 75° flexed. Mean weightbearing ADF was 25° [22°-28°] for the knee extended and 39° [36°-42°] for the knee 75° flexed. The mean differences between 20° knee flexion and full extension were 15° [12°-18°] non weightbearing and 13° [11°-16°] weightbearing. Significant differences of ADF were only found between full extension and 20° of knee flexion. Further knee flexion did not increase ADF.ConclusionKnee flexion of 20° fully eliminates the ADF restraining effect of the gastrocnemius. This knowledge is essential to design a standardized clinical examination assessing MGT.

Development of an accelerated functional rehabilitation protocol following minimal invasive Achilles tendon repair

AbstractPurpose Surgical repair after acute Achilles tendon rupture leads to lower re-rupture rates than non-surgical treatment. After open repair, early functional rehabilitation improves outcome, but there are risks of infection and poor wound healing. Minimal invasive surgery reduces these risks; however, there are concerns about its stability. Consequently, physicians may have reservations about adopting functional rehabilitation. There is still no consensus about the post-operative treatment after minimal invasive repair. The aim of this study was to define the most effective and safe post-operative rehabilitation protocol following minimal invasive repair.Methods A systematic literature search in Embase, MEDLINE and Cochrane Library for prospective trials reporting on early functional rehabilitation after minimal invasive repair was performed. Seven studies were included.ResultsOne randomized controlled trail, one prospective comparative and five prospective non-comparative studies were identified. Four studies performed full weight bearing, all demonstrating good functional results, an early return to work/sports and high satisfaction. One study allowed early mobilization leading to excellent subjective and objective results. The only randomized controlled trial performed the most accelerated protocol demonstrating a superior functional outcome and fewer complications after immediate full weight bearing combined with free ankle mobilization. The non-comparative study reported high satisfaction, good functional results and an early return to work/sports following combined treatment.Conclusion Immediate weight bearing in a functional brace, together with early mobilization, is safe and has superior outcome following minimally invasive repair of Achilles tendon rupture. Our recommended treatment protocol provides quality assurance for the patient and reliability for the attending physician.Level of evidenceII.

Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.

Study protocol: the effect of whole body vibration on acute unilateral unstable lateral ankle sprain- a biphasic randomized controlled trial

BackgroundAnkle sprains often result in ankle instability, which is most likely caused by damage to passive structures and neuromuscular impairment. Whole body vibration (WBV) is a neuromuscular training method improving those impaired neurologic parameters. The aim of this study is to compare the current gold standard functional treatment to functional treatment plus WBV in patients with acute unilateral unstable inversion ankle sprains.Methods/Design60 patients, aged 18–40 years, presenting with an isolated, unilateral, acute unstable inversion ankle sprain will be included in this bicentric, biphasic, randomized controlled trial. Samples will be randomized by envelope drawing. All patients will be allowed early mobilization and pain-dependent weight bearing, limited functional immobilization by orthosis, PRICE, NSARDs as well as home and supervised physiotherapy. Supervised physical therapy will take place twice a week, for 30 minutes for a period of 6 weeks, following a standardized intervention protocol. During supervised physical therapy, the intervention group will perform exercises similar to those of the control group, on a side-alternating sinusoidal vibration platform. Two time-dependent primary outcome parameters will be assessed: short-term outcome after six weeks will be postural control quantified by the sway index; mid-term outcome after one year will be assessed by subjective instability, defined by the presence of giving-way attacks. Secondary outcome parameters include: return to pre-injury level of activities, residual pain, recurrence, objective instability, energy/coordination, Foot and Ankle Disability Index and EQ 5D.DiscussionThis is the first trial investigating the effects of WBV in patients with acute soft tissue injury. Inversion ankle sprains often result in ankle instability, which is most likely due to damage of neurological structures. Due to its unique, frequency dependent, influence on various neuromuscular parameters, WBV is a promising treatment method for patients with acute unstable inversion ankle sprains.Trial registrationNCT01702597