Cornelia Neidlinger-Wilke

Effects of Mechanical Factors on the Fracture Healing Process

An interdisciplinary study based on animal experiments, cell culture studies, and finite element models is presented. In a sheep model, the influence of the osteotomy gap size and interfragmentary motion on the healing success was investigated. Increasing gap sizes delayed the healing process. Increasing movement stimulated callus formation but not tissue quality. Typical distributions of intramembranous bone, endochondral ossification, and connective tissue in the fracture gap are quantified. The comparison of the mechanical data determined by a finite element model with the histologic images allowed the attribution of certain mechanical conditions to the type of tissue differentiation. Intramembranous bone formation was found for strains smaller than approximately 5% and small hydrostatic pressure (<0.15 MPa). Strains less than 15% and hydrostatic pressure more than 0.15 MPa stimulated endochondral ossification. Larger strains led to connective tissue. Cell culture studies on the influence of strain on osteoblasts supported these findings. Proliferation and transforming growth factor beta production was increased for strains up to 5% but decreased for larger strains. Osteoblasts under larger strains (>4%) turned away from the principal strain axis and avoided larger deformations. It is hypothesized that gap size and the amount of strain and hydrostatic pressure along the calcified surface in the fracture gap are the fundamental mechanical factors involved in bone healing.

Cell alignment is induced by cyclic changes in cell length: studies of cells grown in cyclically stretched substrates

Many types of cells, when grown on the surface of a cyclically stretched substrate, align away from the stretch direction. Although cell alignment has been described as an avoidance response to stretch, the specific deformation signal that causes a cell population to become aligned has not been identified. Planar surface deformation is characterized by three strains: two normal strains describe the length changes of two initially perpendicular lines and one shear strain describes the change in the angle between the two lines. The present study was designed to determine which, if any, of the three strains was the signal for cell alignment. Human fibroblasts and osteoblasts were grown in deformable, rectangular, silicone culture dishes coated with ProNectin, a biosynthetic polymer containing the RGD ligand of fibronectin. 24 h after plating the cells, the dishes were cyclically stretched at 1 Hz to peak dish stretches of 0% (control), 4%, 8%, and 12%. After 24 h of stretching, the cells were fixed, stained, and their orientations measured. The cell orientation distribution was determined by calculating the percent of cells whose orientation was within each of eighteen 5° angular intervals. We found that the alignment response was primarily driven by the substrate strain which tended to lengthen the cell (axial strain). We also found that for each cell type there was an axial strain limit above which few cells were found. The axial strain limit for fibroblasts, 4.2 ± 0.4% (mean ± 95% confidence), was lower than for osteoblasts, 6.4 ± 0.6%. We suggest that the fibroblasts are more responsive to stretch because of their more highly developed actin cytoskeleton.

Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity

The cell activity of human-bone-derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human-bone-derived cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 microstrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human-cancellous bone-derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25 and 5-32%, respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

Proliferation of human-derived osteoblast-like cells depends on the cycle number and frequency of uniaxial strain

We tested the hypothesis whether the number of applied load cycles and the frequency of uniaxial strain have an effect on proliferation of human bone derived osteoblast-like cells. A new approach was developed in order to differentiate between the effects of frequency and the effects of cycle number and strain duration. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along there longitudinal axes. Strain was applied over 2 days varying the number of applied load cycles (4-3600) at a constant frequency (1Hz) or varying the frequency (0.1-30Hz) at a constant number of applied cycles (1800) or at a constant strain duration (5min). At a constant frequency, proliferative response increases (103%) with the number of applied cycles until a cycle number maximum (1800 cycles) was reached. 3600 cycles reduced cell number (43%) in contrast to the maximum. The variation of the frequency of applied strain tended to result in slight differences with regard to cell proliferation when cycle number was left constant. However, combined with an appropriate number of cycles there was an optimal frequency (1Hz) as stimulus for bone cell proliferation (84%). A higher frequency (30Hz) in combination with a high cycle number (9000) reduced cell number to control level (4%). This study demonstrates a frequency and cycle number dependent proliferative response of human osteoblast-like cells. It could be shown that effects of the frequency should not be considered separately from the effects of the cycle number.

Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGFβ-release in response to cyclic strain

Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGF beta production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 +/- 6 yr) and 8 normal donors (average age 54 +/- 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGF beta release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGF beta secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGF beta release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts. These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group. While osteoblasts from osteoporotic donors failed to increase proliferation and TGF beta release under the chosen mechanical strain regimen that stimulated both parameters in normal osteoblasts, it is possible that some other strain regimen would provide more effective stimulation of osteoporotic cells.

Behavior of Mesenchymal Stem Cells in the Chemical Microenvironment of the Intervertebral Disc

Study Design. Responses of mesenchymal stem cells (MSCs) from 2 age groups was analyzed under chemical conditions representative of the intervertebral disc (IVD) (low glucose levels, acidic pH, high osmolarity, and combined conditions). Objective. To determine the microenvironmental conditions of the IVD that are critical for MSC-based tissue repair and to determine whether MSCs from different age groups respond differently. Summary of Background Data. MSCs offer promise for IVD repair, but their potential is limited by the harsh chemical microenvironment in which they must survive. Methods. MSCs were isolated from bone marrow from mature (4–5 month old) and young (1 month old) rats and cultured in monolayer under IVD-like glucose, osmolarity, and pH conditions as well as under a combination of these conditions and under standard media conditions for 2 weeks. The response of MSCs was examined by measuring gene expression (real-time RT-PCR), proliferation (MTT assay), and viability (fluorescence staining). Results. Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen-1 expression and caused a small increase in proliferation. In contrast, IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions strongly decreased proliferation and expression of matrix proteins, with more pronounced effects for osmolarity. Combining these 3 conditions also resulted in decreased proliferation, and gene expression of matrix proteins, demonstrating that osmolarity and pH dominated the effects of glucose. Both age groups showed a similar response pattern to the disc microenvironment. Conclusion. IVD repair using MSCs requires increased knowledge of MSC response to the chemical microenvironment. IVD-like low glucose enhanced matrix biosynthesis and maintained cell proliferation whereas IVD-like high osmolarity and low pH conditions were critical factors that reduced biosynthesis and proliferation of young and mature MSCs. Since osmolarity decreases and acidity increases during degeneration, we speculate that pH may be the major limitation for MSC-based IVD repair.

Fibroblast orientation to stretch begins within three hours

Most connective tissue cells align in response to stretch. Previous studies have shown these responses occur within 12–14 h of initiation of stretch, but do not identify the time at which this orientation occurs, nor whether the orientation continues after cessation of stretch. To ascertain the earliest times at which fibroblast orientation occurs, we cultured primary human fibroblasts on deformable culture dishes and stretched (1 Hz, 8% uniaxial strain) them for up to 24 h. We photographed the cells at 0.5, 1–6, 8, 10, 12, 14, 16, and 24 h. Similarly cells were photographed at 1–3, or 4 h after cessation of stretch for stretch durations of 1, 2, and 3 h. Orientation of cells were ascertained by an interactive computer program. The fibroblasts began to orient by 2–3 h and orientation appeared nearly complete by 24 h. Cultures stretched for 2 or 3 h continued to exhibit greater degrees of orientation (compared to controls) for 2 or 3 h respectively after cessation of stretch. We conclude fibroblasts begin to orient within 3 h of initiation of stretch, and that they continue to orient for several hours after cessation of stretch.

Mechanical loading of the intervertebral disc: From the macroscopic to the cellular level

PurposeMechanical loading represents an integral part of intervertebral disc (IVD) homeostasis. This review aims to summarise recent knowledge on the effects of mechanical loads on the IVD and the disc cells, taking into consideration the changes that IVDs undergo during ageing and degeneration, from the macroscopic to the cellular and subcellular level.MethodsNon-systematic literature review.ResultsSeveral scientific papers investigated the external loads that act on the spine and the resulting stresses inside the IVD, which contribute to estimate the mechanical stimuli that influence the cells that are embedded within the disc matrix. As disc cell responses are also influenced by their biochemical environment, recent papers addressed the role that degradation pathways play in the regulation of (1) cell viability, proliferation and differentiation and (2) matrix production and turnover. Special emphasis was put on the intracellular-signalling pathways, as mechanotransduction pathways play an important role in the maintenance of normal disc metabolism and in disc degenerative pathways.ConclusionsDisc cells are exposed to a wide range of mechanical loads, and the biochemical environment influences their responses. Degeneration-associated alterations of the disc matrix change the biochemical environment of disc cells and also the mechanical properties of the disc matrix. Recent studies indicate that these factors interact and regulate disc matrix turnover.

Mitogens are increased in the systemic circulation during bone callus healing

The influence of mechanical tissue strain caused by flexible fracture fixation on the systemic occurrence of systemic mitogens during callus healing was investigated. For this purpose the mitogenic capacity and growth factor concentration of sera from patients undergoing fracture treatment were determined. Sera from 9 patients whose fractures had been stabilized by external fixation were collected before and during fracture treatment. The sera were added to cell culture media of the osteoblastic cell line SaOS‐2. After 5‐6 days cell proliferation was measured. Transforming growth factor‐β1 (TGF‐β1) and insulin‐like growth factor‐I (IGF‐I) concentrations were analyzed in serum samples from different healing stages. Statistics: paired Wilcoxon‐test. Sera from fracture patients decreased SaOS‐2 proliferation in the first week after surgery (p<0.05) compared to sera obtained prior to surgery. In the fourth or fifth week proliferation increased significantly (p<0.03). The increased proliferation of the SaOS‐2 cells was associated with elevated levels of TGF‐β and IGF‐I (p<0.05). The higher mitogenic activity of sera suggests an increased level of circulating mitogens. In a previous study this increase had also been observed in patients during distraction osteogenesis treatment but not in patients with primary bone healing by a stable fixated plate. It is therefore assumed that their release from the fracture site is a consequence of mechanical stimulation by interfragmentary movement of fracture ends.

Cell sources for nucleus pulposus regeneration

PurposeThere is increasing interest in the development of cell therapy as a possible approach for the treatment of degenerative disc disease. To regenerate nucleus pulposus tissue, the cells must produce an appropriate proteoglycan-rich matrix, as this is essential for the functioning of the intervertebral disc. The natural environment within the disc is very challenging to implanted cells, particularly if they have been subcultured in normal laboratory conditions. The purpose of this work is to discuss parameters relevant to translating different proposed cell therapies of IVD into clinical use.ResultsSeveral sources of cells have been proposed, including nucleus pulposus cells, chondrocytes and mesenchymal stem cells derived from bone marrow or adipose tissue. There are some clinical trials and reports of attempts to regenerate nucleus pulposus utilising either autologous or allogenic cells. While the published results of clinical applications of these cell therapies do not indicate any safety issues, additional evidence will be needed to prove their long-term efficacy.ConclusionThis article discusses parameters relevant for successful translation of research on different cell sources into clinically applicable cell therapies: the influence of the intervertebral disc microenvironment on the cell phenotype, issues associated with cell culture and technical preparation of cell products, as well as discussing current regulatory requirements. There are advantages and disadvantages of each proposed cell type, but no strong evidence to favour any one particular cell source at the moment.