Limitations and challenges in MSC delivery for bone regeneration

Objective: Regeneration of osseous defects by tissue-engineering or cell delivery approach provides a novel means of treatment utilizing cell biology, materials sciences, and molecular biology. The concept of in vitro explanted mesenchymal stem cells (MSCs) with an ability to induce new bone formation has been demonstrated in some small animal models. However, contradictory results have been reported regarding the regenerative capacity of MSCs after ex vivo expansion due to the lack of the understanding of microenvironment for MSC differentiation in vivo. ----- ----- Methods: In our laboratory tissue-derived and bone marrow-derived MSCs have been investigated in their osteogenesis. Cell morphology and proliferation were studied by microscopy, confocal microscopy, FACS and cell counting. Cell differentiation and matrix formation were analysed by matrix staining, quantitative PCR, and immunohistochemistry. A SCID skull defect model was used for cell transplantation studies.----- ----- Results: It was noted that tissue-derived and bone marrow-derived MSCs showed similar characteristics in cell surface marker expression, mesenchymal lineage differentiation potential, and cell population doubling. MSCs from both sources could initiate new bone formation in bone defects after delivery into a critical size defects. The bone forming cells were from both transplanted cells and endogenous cells from the host. Interestingly, the majority of in vitro osteogenic differentiated cells did not form new bone directly even though mineralized matrix was synthesized in vitro by MSCs. Furthermore, no new bone formation was detected when MSCs were transplanted subcutaneously.----- ----- Conclusion: This study unveiled the limitations of MSC delivery in bone regeneration and proposed that in vivo microenvironment needs to be optimized for MSC delivery in osteogenesis.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

Mechanical conditions in the initial phase of bone healing

Background: Bone healing is sensitive to the initial mechanical conditions with tissue differentiation being determined within days of trauma. Whilst axial compression is regarded as stimulatory, the role of interfragmentary shear is controversial. The purpose of this study was to determine how the initial mechanical conditions produced by interfragmentary shear and torsion differ from those produced by axial compressive movements. ----- ----- Methods: The finite element method was used to estimate the strain, pressure and fluid flow in the early callus tissue produced by the different modes of interfragmentary movement found in vivo. Additionally, tissue formation was predicted according to three principally different mechanobiological theories. ----- ----- Findings: Large interfragmentary shear movements produced comparable strains and less fluid flow and pressure than moderate axial interfragmentary movements. Additionally, combined axial and shear movements did not result in overall increases in the strains and the strain magnitudes were similar to those produced by axial movements alone. Only when axial movements where applied did the non-distortional component of the pressure–deformation theory influence the initial tissue predictions. ----- ----- Interpretation: This study found that the mechanical stimuli generated by interfragmentary shear and torsion differed from those produced by axial interfragmentary movements. The initial tissue formation as predicted by the mechanobiological theories was dominated by the deformation stimulus.

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing : a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.

Temporal tissue patterns in bone healing of sheep

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Spatial and temporal variations of mechanical properties and mineral content of the external callus during bone healing

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing - An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Osteoclastic activity begins early and increases over the course of bone healing

Osteoclasts are specialised bone-resorbing cells. This particular ability makes osteoclasts irreplaceable for the continual physiological process of bone remodelling as well as for the repair process during bone healing. Whereas the effects of systemic diseases on osteoclasts have been described by many authors, the spatial and temporal distribution of osteoclasts during bone healing seems to be unclear so far. In the present study, healing of a tibial osteotomy under standardised external fixation was examined after 2, 3, 6 and 9 weeks (n = 8) in sheep. The osteoclastic number was counted, the area of mineralised bone tissue was measured histomorphometrically and density of osteoclasts per square millimetre mineralised tissue was calculated. The osteoclastic density in the endosteal region increased, whereas the density in the periosteal region remained relatively constant. The density of osteoclasts within the cortical bone increased slightly over the first 6 weeks, however, there was a more rapid increase between the sixth and ninth weeks. The findings of this study imply that remodelling and resorption take place already in the very early phase of bone healing. The most frequent remodelling process can be found in the periosteal callus, emphasising its role as the main stabiliser. The endosteal space undergoes resorption in order to recanalise the medullary cavity, a process also started in the very early phase of healing at a low level and increasing significantly during healing. The cortical bone adapts in its outward appearance to the surrounding callus structure. This paradoxic loosening is caused by the continually increasing number and density of osteoclasts in the cortical bone ends. This study clearly emphasises the osteoclastic role especially during early bone healing. These cells do not simply resorb bone but participate in a fine adjusted system with the bone-producing osteoblasts in order to maintain and improve the structural strength of bone tissue.

Endochondral ossification in vitro is influenced by mechanical bending

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

CYR61 (CCN1) protein expression during fracture healing in an ovine tibial model and its relation to the mechanical fixation stability

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

The course of bone healing is influenced by the initial shear fixation stability

Fracture healing is influenced by fixation stability and experimental evidence suggests that the initial mechanical conditions may determine the healing outcome. We hypothesised that mechanical conditions influence not only the healing outcome, but also the early phase of fracture healing. Additionally, it was hypothesised that decreased fixation stability characterised by an increased shear interfragmentary movement results in a delay in healing. Sixty-four sheep underwent a mid-shaft tibial osteotomy which was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture callus was analysed using radiological, biomechanical and histological techniques. The tibia treated with semi-rigid fixation showed inferior callus stiffness and quality after 6 weeks. At 9 weeks, the calluses were no longer distinguishable in their mechanical competence. The calluses at 9 weeks produced under rigid fixation were smaller and consisted of a reduced fibrous tissue component. These results demonstrate that the callus formation over the course of healing differed both morphologically and in the rate of development. In this study, we provide evidence that the course of healing is influenced by the initial fixation stability. The semi-rigid fixator did not result in delayed healing, but a less optimal healing path was taken. An upper limit of stability required for successful healing remains unknown, however a limit by which healing is less optimal has been determined.

Addressing sustainable living using a collaborative approach and multi-disciplinary techniques

In order to achieve meaningful reductions in individual ecological footprints, individuals must dramatically alter their day to day behaviours. Effective interventions will need to be evidence based and there is a necessity for the rapid transfer or communication of information from the point of research, into policy and practice. A number of health disciplines, including psychology and public health, share a common mission to promote health and well-being and it is becoming clear that the most practical pathway to achieving this mission is through interdisciplinary collaboration. This paper argues that an interdisciplinary collaborative approach will facilitate research that results in the rapid transfer of findings into policy and practice. The application of this approach is described in relation to the Green Living project which explored the psycho-social predictors of environmentally friendly behaviour. Following a qualitative pilot study, and in consultation with an expert panel comprising academics, industry professionals and government representatives, a self-administered mail survey was distributed to a random sample of 3000 residents of Brisbane and Moreton Bay (Queensland, Australia). The Green Living survey explored specific beliefs which included attitudes, norms, perceived control, intention and behaviour, as well as a number of other constructs such as environmental concern and altruism. This research has two beneficial outcomes. First, it will inform a practical model for predicting sustainable living behaviours and a number of local councils have already expressed an interest in making use of the results as part of their ongoing community engagement programs. Second, it provides an example of how a collaborative interdisciplinary project can provide a more comprehensive approach to research than can be accomplished by a single disciplinary project.

Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration