Comparison of biophysical stimuli for mechano-regulation of tissue differentiation during fracture healing

Most long-bone fractures heal through indirect or secondary fracture healing, a complex process in which endochondral ossification is an essential part and bone is regenerated by tissue differentiation. This process is sensitive to the mechanical environment, and several authors have proposed mechano-regulation algorithms to describe it using strain, pore pressure and/or interstitial fluid velocity as biofeedback variables. The aim of this study was to compare various mechano-regulation algorithms' abilities to describe normal fracture healing in one computational model. Additionally, we hypothesized that tissue differentiation during normal fracture healing could be equally well regulated by the individual mechanical stimuli, e.g. deviatoric strain, pore pressure or fluid velocity. A biphasic finite element model of an ovine tibia with a 3mm fracture gap and callus was used to simulate the course of tissue differentiation during normal fracture healing. The load applied was regulated in a biofeedback loop, where the load magnitude was determined by the interfragmentary movement in the fracture gap. All the previously published mechano-regulation algorithms studied, simulated the course of normal fracture healing correctly. They predicted (1) intramembranous bone formation along the periosteum and callus tip, (2) endochondral ossification within the external callus and cortical gap, and (3) creeping substitution of bone towards the gap from the initial lateral osseous bridge. Some differences between the effects of the algorithms were seen, but they were not significant. None of the volumetric components, i.e. pore pressure or fluid velocity, alone were able to correctly predict spatial or temporal tissue distribution during fracture healing. However, simulation as a function of only deviatoric strain accurately predicted the course of normal fracture healing. This suggests that the deviatoric component may be the most significant mechanical parameter to guide tissue differentiation during indirect fracture healing.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13-/- mice is intrinsic to cartilage and bone. Mmp13-/- mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Site specific effects of zoledronic acid during tibial and mandibular fracture repair

Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones.     

A computational model to explore the role of angiogenic impairment on endochondral ossification during fracture healing

While it is well established that an adequate blood supply is critical to successful bone regeneration, it remains poorly understood how progenitor cell fate is affected by the altered conditions present in fractures with disrupted vasculature. In this study, computational models were used to explore how angiogenic impairment impacts oxygen availability within a fracture callus and hence regulates mesenchymal stem cell (MSC) differentiation and bone regeneration. Tissue differentiation was predicted using a previously developed algorithm which assumed that MSC fate is governed by oxygen tension and substrate stiffness. This model was updated based on the hypothesis that cell death, chondrocyte hypertrophy and endochondral ossification are regulated by oxygen availability. To test this, the updated model was used to simulate the time course of normal fracture healing, where it successfully predicted the observed quantity and spatial distribution of bone and cartilage at 10 and 20 days post-fracture (dpf). It also predicted the ratio of cartilage which had become hypertrophic at 10 dpf. Following this, three models of fracture healing with increasing levels of angiogenic impairment were developed. Under mild impairment, the model predicted experimentally observed reductions in hypertrophic cartilage at 10 dpf as well as the persistence of cartilage at 20 dpf. Models of more severe impairment predicted apoptosis and the development of fibrous tissue. These results provide insight into how factors specific to an ischemic callus regulate tissue regeneration and provide support for the hypothesis that chondrocyte hypertrophy and endochondral ossification during tissue regeneration are inhibited by low oxygen.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens

Summary The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present.The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens.In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens.The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. II. The glycosaminoglycans

Summary The glycosaminoglycans secreted into the matrices associated with fractures of the rabbit tibia healing under stable and unstable mechanical conditions have been characterized histochemically using the dye Alcian Blue at pH 5.7 in the presence of increasing concentrations of magnesium chloride, and after enzymatic extractions. These results are compared with those of immunohistochemical experiments using monoclonal antibodies which recognize epitopes specific to various glycosaminoglycans.The results indicate that the fibrous tissues, including those of the cavities of the cancellous bone and periosteum, possess hyaluronate and chondroitin sulphate, but the amounts present are small. The glycosaminoglycans detected in the cortical bone are located mainly around the osteocyte lacunae where chondroitin and keratan sulphates are found. The developing trabeculae of cancellous bone in the callus contain chondroitin and keratan sulphates, but as the trabeculae mature, these glycosaminoglycans are no longer present throughout the matrix; they are found particularly around the osteocyte lacunae.The cartilage in the callus of mechanically unstable fractures contains chondroitin, chondroitin-4- and 6-sulphates and keratan sulphate, though their distribution is variable. The small, transient areas of cartilage in the callus of mechanically stable fractures also contain those glycosaminoglycans, but they appear to be less highly sulphated.The mechanical stability of the fractures appears to affect the amount and degree of sulphation of the glycosaminoglycans, rather than the types of glycosaminoglycan produced. The glycosaminoglycans produced during fracture healing are compared with those produced during embryonic development and other healing processes.     

Mepe is expressed during skeletal development and regeneration

Matrix extracellular phosphoglycoprotein (Mepe) is a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.     

Type X collagen synthesis during endochondral ossification in fracture repair

Collagen synthesis in normal connective tissue development and repair is integral to tissue stability. The appearance of a short chain collagen, designated Type X, was studied in experimental fractures created in the chicken humerus. Biosynthetic studies using [14C]proline incorporation coupled with histologic examination of the cartilaginous callus demonstrated that Type X collagen synthesis occurs during endochondral ossification in the fracture callus. Type X synthesis occurred in the areas of cartilaginous callus composed of hypertrophic and degenerative chondrocytes that were associated with increased vascularity and matrix mineralization. Synthesis of short chain collagen was not detected in either skeletal muscle or bone. Two-dimensional peptide mapping of cyanogen bromide and proteolytic fragments derived from fracture callus short chain collagen confirmed the identity of this collagen as Type X. The synthesis of Type X collagen by fracture callus is further evidence supporting its close association with the process of endochondral ossification.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification

ObjectiveSensory and sympathetic nerve fibers (SNF) innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of the absence of sensory neurotransmitter substance P (SP) and the removal of SNF on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation.MethodsIn order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1 −/−) mice (no SP) and animals without SNF. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and β2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2, and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts, and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all the three groups for mechanical testing and μCT-analysis.ResultsCallus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained β2-adrenoceptor and TH-negative. Absence of SP and SNF did not change the general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5 days after fracture. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1 −/− mice and mice without SNF, a lower number of osteoblasts in Tac1 −/− mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of osteoclasts and osteoblasts isolated from Tac1 −/− and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21 days after fracture, revealed an overall reduced mechanical bone quality in Tac1 −/− mice and mice without SNF. μCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF.ConclusionThe absence of SP and SNF reduces pain sensitivity and mechanical stability of the bone in general. The micro-architecture of the bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, the absence of SP reduces bone formation rate whereas the absence of SNF induces bone resorption rate. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.     

Three-dimensional reconstruction of fracture callus morphogenesis.

Rat and mouse femur and tibia fracture calluses were collected over various time increments of healing. Serial sections were produced at spatial segments across the fracture callus. Standard histological methods and in situ hybridization to col1a1 and col2a1 mRNAs were used to define areas of cartilage and bone formation as well as tissue areas undergoing remodeling. Computer-assisted reconstructions of histological sections were used to generate three-dimensional images of the spatial morphogenesis of the fracture calluses. Endochondral bone formation occurred in an asymmetrical manner in both the femur and tibia, with cartilage tissues seen primarily proximal or distal to the fractures in the respective calluses of these bones. Remodeling of the calcified cartilage proceeded from the edges of the callus inward toward the fracture producing an inner-supporting trabecular structure over which a thin outer cortical shell forms. These data suggest that the specific developmental mechanisms that control the asymmetrical pattern of endochondral bone formation in fracture healing recapitulated the original asymmetry of development of a given bone because femur and tibia grow predominantly from their respective distal and proximal physis. These data further show that remodeling of the calcified cartilage produces a trabecular bone structure unique to fracture healing that provides the rapid regain in weight-bearing capacity to the injured bone.     

Identification of tissue differentiation rates in a mechanobiological model of fracture healing

As a basis for model-based analysis of the processes in secondary fracture healing, a dynamical model is presented that characterises the physiological status in the fracture area by the location-dependent composition of tissues. Five types of tissue are distinguished: connective tissue, cartilage, bone, haematoma and avascular bone. A rule base is given that describes dynamical tissue differentiation processes. The rules consider not only a mechanical stimulus but also osteogenic and a vasculative factors as biological stimuli. Within this model structure, it is possible, e.g., to distinguish intramembranous from endochondral ossification processes. An objective function is introduced to assess accordance between the model-based simulation results and reference healing stages. By minimising this objective function, relevant tissue differentiation rates can be determined. For a reference process of secondary fracture healing it could be shown that the intramembranous ossification rate of 0.313%/day (from connective tissue to bone) is much smaller than the endochondral ossification rate of 1.136%/day (from cartilage to bone). In order to verify the model approach, it is transferred to simulate long bone distraction. Results show that healing patterns of bone distraction can be predicted. Using this method, it is possible to identify model parameters for individual subjects. This will allow a patient-specific analysis of tissue healing processes in future.     

Trabecular bone fracture healing simulation with finite element analysis and fuzzy logic

Trabecular bone fractures heal through intramembraneous ossification. This process differs from diaphyseal fracture healing in that the trabecular marrow provides a rich vascular supply to the healing bone, there is very little callus formation, woven bone forms directly without a cartilage intermediary, and the woven bone is remodelled to form trabecular bone. Previous studies have used numerical methods to simulate diaphyseal fracture healing or bone remodelling, however not trabecular fracture healing, which involves both tissue differentiation and trabecular formation. The objective of this study was to determine if intramembraneous bone formation and remodelling during trabecular bone fracture healing could be simulated using the same mechanobiological principles as those proposed for diaphyseal fracture healing. Using finite element analysis and the fuzzy logic for diaphyseal healing, the model simulated formation of woven bone in the fracture gap and subsequent remodelling of the bone to form trabecular bone. We also demonstrated that the trabecular structure is dependent on the applied loading conditions. A single model that can simulate bone healing and remodelling may prove to be a useful tool in predicting musculoskeletal tissue differentiation in different vascular and mechanical environments.     

Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1(-/-) calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.     

Modelling the effects of different fracture geometries and healing stages on ultrasound signal loss across a long bone fracture

The effect on the signal amplitude of ultrasonic waves propagating along cortical bone plates was modelled using a 2D Finite Difference code. Different healing stages, represented by modified fracture geometries were introduced to the plate model. A simple transverse and oblique fracture filled with water was introduced to simulate the inflammatory stage. Subsequently, a symmetric external callus surrounding a transverse fracture was modelled to represent an advanced stage of healing. In comparison to the baseline (intact plate) data, a large net loss in signal amplitude was produced for the simple transverse and oblique cases. Changing the geometry to an external callus with different mechanical properties caused the net loss in signal amplitude to reduce significantly. This relative change in signal amplitude as the geometry and mechanical properties of the fracture site change could potentially be used to monitor the healing process.     

Modelling the effects of different fracture geometries and healing stages on ultrasound signal loss across a long bone fracture

The effect on the signal amplitude of ultrasonic waves propagating along cortical bone plates was modelled using a 2D Finite Difference code. Different healing stages, represented by modified fracture geometries were introduced to the plate model. A simple transverse and oblique fracture filled with water was introduced to simulate the inflammatory stage. Subsequently, a symmetric external callus surrounding a transverse fracture was modelled to represent an advanced stage of healing. In comparison to the baseline (intact plate) data, a large net loss in signal amplitude was produced for the simple transverse and oblique cases. Changing the geometry to an external callus with different mechanical properties caused the net loss in signal amplitude to reduce significantly. This relative change in signal amplitude as the geometry and mechanical properties of the fracture site change could potentially be used to monitor the healing process.     

Low-Dose X-Ray Irradiation Promotes Osteoblast Proliferation,Differentiation and Fracture Healing

Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy) X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy) X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.