Callus Size Research Articles

Comment on permeability conditions in finite element simulation of bone fracture healing

The most popular model of the bone healing considers the fracture callus as poroelastic medium. As such it requires an assumption of the callus’ external permeability. In this work a systematic study of the influence of the permeability of the callus boundary on the simulated bone healing progress is performed. The results show, that these conditions starts to play significant role with the decrease of the callus size. Typically enforced impermeability inhibits the progress of healing during simulation. A remedy for this effect is imposing drainage conditions at the callus’ boundary.

Essential oil constituents and secondary metabolites of Mentha viridis under tissue culture technique using violet visible light emitting diodes (LEDs)

This study aimed to propagate the valuable medicinal plant Mentha viridis through in vitro culture of nodal segments measuring approximately 1-1.5 cm. Two different types of light-emitting diode (LED) systems were used to apply three different concentrations of two different cytokinins: 6-benzylaminopurine (BAP) and thidiazuron (TDZ) at 0, 1, or 2 mg/L. The LED systems were white as a control and violet, which is a 1:1 ratio of red and blue light. After a 30-day incubation period, the results revealed significant improvements in the survival rate and the number of shoots per explant across the various treatment groups. With MS medium supplemented with 2 mg/L TDZ and illuminated by white and violet LEDs, the highest values were obtained, yielding survival rates of 93.3% and 13.3 shoots per explant, respectively. Moreover, the treatment involving 2 mg/L TDZ under violet LEDs illumination exhibited superior outcomes in terms of leaf count per explant, callus formation, and callus size. Notably, no callus formation was observed in response to BAP treatments. All treatments resulted in a significant increase in antioxidant enzyme activity and the accumulation of various compounds, such as anthocyanin, ascorbic acid, phenols, flavonoids, peroxidase, and polyphenol oxidase, when compared to the control in a broader context, the levels of IAA, kinetin, and zeatin increased, while GA3 and ABA decreased in response to the applied treatments, as compared to the control. Additionally, ten compounds were consistently found in all treatments by GC/MS analysis of the micro-propagated Mentha, with carvone accounting for the highest proportion (43.5%) and being the predominant component. Among all treatments, nodal segments that were exposed to violet LEDs and grown on MS medium supplemented with 1 mg/L TDZ had the highest carvone content.

THERAPEUTIC OPTIONS IN CALCIFIC ROTATOR CUFF TENDINOPATHY OF THE SHOULDER

Calcific tendinopathy in the tendons of the rotator cuff muscles of the shoulder is an increasingly common and disabling pathology. Calcific tendinopathy is a common disease that mainly affects people aged between 40 and 60. Women seem to be more affected than men. It has been suggested that various factors play a role in the condition, such as abnormal thyroid gland activity, metabolic diseases (e.g. gout, diabetes) and genetic predisposition. Various aetiological hypotheses have been put forward: the degenerative and multiphasic theories are the two most widely accepted. Clinically, calcific tendonitis is characterised by severe, disabling pain, often occurring spontaneously, usually in the morning or following exertion disproportionate to subsequent symptoms. There may be concomitant stiffness, giving rise to a clinical picture resembling frozen shoulder or hyperalgesic painful shoulder. Conventional radiography of the shoulder is the most appropriate imaging approach, but perfectly complemented by soft tissue ultrasound. Most acute painful episodes resolve spontaneously symptomatically. Numerous conservative treatments have been reported in the literature with varying levels of evidence of efficacy. In the present study we tried to evaluate which therapeutic option could give superior results both from a functional, lesional architectural and causal point of view in terms of reduction of callus size, but also from an imaging point of view. The use of various therapeutic protocols have shown a significant benefit of the association of ESWT with conventional FKT, and intra-articular infiltration, with a plus for the association of Corticosteroids and Sodium Hyaluronate regarding the speed of achieving the desired results. Similar results were obtained with the intra-articular shoulder PRP technique, but with slower achievement of the desired results. Arthroscopic surgery is the orthopaedic specialist's last option. Keywords: Calcific tendinopathy, rotator cuff muscles, shoulder, intra-articular infiltration.

The Effect of Some Plant Growth Regulators on Callus Culture of Different Pistachio Varieties

Pistachio (Pistacia vera L) is one of the oldest cultivated plants in the world. Its fruits are rich in protein, minerals, carbohydrates, fiber, and vitamins. In addition, the demand for these plants is increasing due to the fact that they are very tasty and nutritious. On the other hand, pistachio cultivation is quite difficult. In addition, many problems are encountered in germination with seeds or reproduction with cuttings. These situations necessitate the development of different in vitro tissue culture protocols. In this study, callus culture optimization protocol was developed by using seeds of three different pistachio cultivars. Murashige and Skoog (MS) medium was supplemented with different concentrations of NAA, IAA, 2,4 D and BAP. When callus size (1,776 cm), callus weight (0.908 g) and embryogenic callus regenerations (27.94%) were considered, it was found that the best variety was Tekin. Again, in the evaluation made according to these factors, it was determined that the best improvement was in the MS medium containing 3 mg/L BAP and 1 mg/L 2,4D. The contamination rate detected throughout the studies ranged from 7.65% to 12.91%.

Pengaruh waktu induksi dan ZPT terhadap pertumbuhan kalus kotiledon kacang panjang (Vigna unguiculata subsp. sesquipedalis)

Yard-Long bean plants are an agricultural commodity that has great potential to be developed because they are easy to cultivate, their market share is also quite high but there are still problems in meeting the need for sufficient seeds and they are still susceptible to pests and disease. Therefore, efforts need to be made to increase production and induce variations. One method that can be used is tissue culture for the induction of somaclonal variations and/or the formation of transgenic plants. This study aims to determine the effect of variations in callus initiation time and the effect of Plant Growth Regulators (PGR) administration on the growth of long bean cotyledon explant callus. Seed sterilization was carried out using detergent, 10% sodium hypochlorite, 70% alcohol and sterile distilled water. The results of the research showed that variations in callus initiation time on MS media with the addition of 2,4-D PGR and the use of several variations of PGR in subcultures were able to initiate callus and influence callus size, callus color, and the percentage of browning of Yard-long bean cotyledon explants. Callus initiation time of 4 weeks with a combination of 2 ppm BAP concentration with 0.1 ppm IAA produced the largest callus size and brownish white callus color. The addition of PGR during subculture had a smaller browning percentage than the treatment without PGR.

Effect of culture filtrates of Fusarium fungi on oat callus cultures

The effect of three concentrations (30, 40 and 50%) of the culture filtrate (CF) of the genus Fusarium fungi (F. sporotrichioides, F. poae, F. equiseti, F. oxysporum) on the growth and development parameters of oat callus culture was evaluated. CF at the callus proliferation stage was used to select somaclonal cell lines with mycotoxin tolerance traits. Prior to this, callusogenesis induction was carried out on the Murashige-Skoog (MS) medium with 3 mg/l 2,4-D and 2 mg/l UIC in the culture of immature embryos of the Tyumensky Golozerny, Tubinsky, Zolotoy Pochatok and Talisman varieties. The material for the experiment was selected from the ears of the plants grown in the summer period of 2016–2018 on the experimental fields in the Krasnoyarsk forest-steppe. Callus size was recorded when transplanted onto the proliferation media (MS + 1.5 mg/l 2,4-D), control and CF-containing media. After 30 days of cultivation, callus growth, signs of necrosis and organogenesis were noted. At the end of the experiment, the number of the regenerants formed was counted. The presence of CF in the proliferation medium already at a concentration of 40% provided a decrease in the proliferative activity and increased the frequency of necrosis by at least 50%. Similar results were obtained at the CF level of 50%. On the media with F. poae CF, the reduction of callus viability reached 60–70%. The calluses that remained viable under these conditions had a frequency of regenerant formation and organogenesis 2–3 times higher than the samples that were not influenced by the selective factor. This is particularly pronounced when F. sporotrichioides CF is added. However, this effect was not observed when F. oxysporum CF was applied. This may be due to the differences in the composition of the mycotoxin complex of this mushroom species from the others used in the study. For further work on the technology of creating oat forms with resistance to mycotoxins of the Fusarium genus fungi, it is assumed to use a level of selective pressure not lower than 40%.

The HOS1-PIF4/5 module controls callus formation in Arabidopsis leaf explants

ABSTRACT A two-step plant regeneration has been widely exploited to genetic manipulation and genome engineering in plants. Despite technical importance, understanding of molecular mechanism underlying in vitro plant regeneration remains to be fully elucidated. Here, we found that the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1)-PHYTOCHROME INTERACTING FACTOR 4/5 (PIF4/5) module participates in callus formation. Consistent with the repressive role of HOS1 in PIF transcriptional activation activity, hos1–3 mutant leaf explants exhibited enhanced callus formation, whereas pif4–101 pif5–3 mutant leaf explants showed reduced callus size. The HOS1-PIF4/5 function would be largely dependent on auxin biosynthesis and signaling, which are essential for callus initiation and proliferation. Our findings suggest that the HOS1-PIF4/5 module plays a pivotal role in auxin-dependent callus formation in Arabidopsis.

Effects of 2,4-dichlorophenoxyacetic acid, α-naphthylacetic acid on the variant formation of Valentine Hoya (Hoya kerrii)

Valentine Hoya (Hoya kerrii) is favoured by many people for its richness in leaf shape, colour and flower fragrance. To increase the diversity and abundance of Hoya, leaf samples were transplanted into MS medium supplemented with 2,4-D and α-naphthylacetic acid (NAA) (concentration 4-7 mg/l) to create callus and induce mutations. Calli formation rate was observed at 100% after 15 days of culture in all treatments supplemented with growth regulators, while the control treatment showed no callus formation. The results of rapid callus multiplication showed that the callus-forming leaf sample obtained from the treatment of 5 mg/l 2,4-D gave the largest callus size (2.98 cm). The 45-day-old calli were transferred to MS + 1 mg/l IBA + 3.5 mg/l BAP regeneration medium, the time of shoot appearance for callus samples treated with NAA was 95 days after subculture with the highest regeneration rate of 66.67%, earlier than 2,4-D treated calli (225 days, 50% regeneration rate). The results of genetic divergence analysis using RAPD markers recorded a total of 330 DNA bands with molecular sizes ranging from 170 to 2700 bp. The polymorphism information content index was calculated in the 0.23-0.40 range. Samples treated with 5 mg/l NAA had the lowest similarity coefficient of 0.455, growth parameters such as petiole diameter, dermal tissue thickness, vascular bundle area, and a number of wood bundles recorded in samples treated with 5 mg/l NAA were superior to those of the control.Keywords:α-naphthylacetic acid, Hoya kerrii, polymorphisminformation content, variation, 2,4-D.Classification number:4.6

Induction of Ploidy Level on Three Patchouli (Pogostemon cablin Benth) Cultivars by Colchicine In Vitro

Patchouli plants produce essential oils and are used as a raw material and fixative agent for perfumes. The most widely cultivated patchouli plant in Indonesia is Aceh patchouli. Sexual propagation in Aceh patchouli plants is impossible as it does not flower. Genetic diversity can be elevated through another method, such as polyploidy induction using colchicine. This research aimed to evaluate the state-of-the-art application of polyploidization techniques as a breeding tool for callus-based explants. A completely randomized design with a factorial pattern was used in this experiment consisting of two factors, patchouli cultivars (Sidikalang, Tapak Tuan, and Lhokseumawe) and colchicine concentration (0.0%, 0.2%, 0.5%, 0.7%, and 1%). The result showed an interaction between three patchouli cultivars with colchicine concentration on callus size and color characters. In Sidikalang cultivar, 0.2% colchicine concentration affects the character of callus emergence time and has more buds. In cultivar Tapak Tuan, the application of colchicine 0.2% affects the callus size character and has the highest number of buds. In the Lhokseumawe cultivar, giving 0.5% colchicine affects the character of callus appearance time, callus size, and callus color. Concentrations of 0.2% and 1.0% colchicine in Sidikalang and Tapak Tuan calluses increased the number of chromosomes, ranging from 2n (32), 3n (48), and 4n (64). The implication of the research could be disclosure of an opportunity to create a new superior variety.

Histological Examination of the Effects of Thrombopoietin Mimetic Peptide (TMP) and High-Fat Diet on Femur Fracture Healing

Background and Hypothesis: In the US, 11.3% of the population are diabetic. Impaired bone healing is a complication of diabetes that dramatically impacts quality of life. Thus, it is imperative to find effective, low-risk treatments for patients that can accelerate fracture healing. We propose treatment of femur fractures using a thrombopoietin (TPO) analogue, TMP, will expedite healing, reduce adverse side effects compared to FDA-approved BMP-2, and improve quality of life of diabetic fracture patients.
 Experimental Design or Project Methods: Tie2CreERT+ mice were bred with Mplfl/fl mice to generate mice in which the TPO receptor (Mpl) was deleted in cells of the endothelial lineage (Tie2 expressing cells) following tamoxifen induction (3 consecutive daily 10mg/kg doses). Tie2CreERT+ ; Mplfl/fl and Tie2CreERT+ ; Mpl+/+ mice served as experimental and control mice, respectively. Eight-week-old male mice of both genotypes were placed on a low-fat diet (LFD) or high-fat-diet (HFD) for 12 weeks. One week prior to surgery, mice were injected with tamoxifen to induce Cre-recombination. Mice were then subjected to femur fracture and treated with saline or TMP (33nmol/kg/day) for the first week post-surgery. Mice were euthanized at 1-, 2-, and 4-week post-surgery and injured femurs were isolated for histological evaluation of the fracture callus size and composition.
 Results: To date only Tie2CreERT+ ; Mplfl/fl specimens have been processed. As expected, untreated HFD mice exhibited impaired fracture healing compared to similarly untreated LFD mice. As would also be expected, no differences were observed in fracture healing histological parameters between saline and TMP treated Tie2 CreERT+ ; Mplfl/fl mice at similar time points post-surgery.
 Conclusion and Potential Impact: While ongoing, this study explores the efficacy of using thrombopoietic agents for fracture healing in type 2 diabetes. If promising, thrombopoietic agents could replace, BMP-2 treatment, and may improve the quality of life for individuals experiencing impaired fracture healing.

Microgel-Assisted Delivery of Adenosine to Accelerate Fracture Healing.

Extracellular adenosine plays a key role in promoting bone tissue formation. Local delivery of adenosine could be an effective therapeutic strategy to harness the beneficial effect of extracellular adenosine on bone tissue formation following injury. Herein, we describe the development of an injectable in situ curing scaffold containing microgel-based adenosine delivery units. The two-component scaffold includes adenosine-loaded microgels and functionalized hyaluronic acid (HA) molecules. The microgels were generated upon copolymerization of 3-acrylamidophenylboronic acid (3-APBA)- and 2-aminoethylmethacrylamide (2-AEMA)-conjugated HA (HA-AEMA) in an emulsion suspension. The PBA functional groups were used to load the adenosine molecules. Mixing of the microgels with the HA polymers containing clickable groups, dibenzocyclooctyne (DBCO) and azide (HA-DBCO and HA-Azide), resulted in a 3D scaffold embedded with adenosine delivery units. Application of the in situ curing scaffolds containing adenosine-loaded microgels following tibial fracture injury showed improved bone tissue healing in a mouse model as demonstrated by the reduced callus size, higher bone volume, and increased tissue mineral density compared to those treated with the scaffold without adenosine. Overall, our results suggest that local delivery of adenosine could potentially be an effective strategy to promote bone tissue repair.

Optimization of Callus and Cell Suspension Cultures of Lycium schweinfurthii for Improved Production of Phenolics, Flavonoids, and Antioxidant Activity

Lycium schweinfurthii is a traditional medicinal plant grown in the Mediterranean region. As it is used in folk medicine to treat stomach ulcers, it took more attention as a source of valuable secondary metabolites. The in vitro cultures of L. schweinfurthii could be a great tool to produce secondary metabolites at low costs. The presented study aimed to introduce and optimize a protocol for inducing callus and cell suspension cultures as well as estimating phenolic, flavonoid compounds, and antioxidant activity in the cultures of the studied species. Three plant growth regulators (PGRs) were supplemented to MS medium solely or in combination to induce callus from leaf explants. The combination between 2,4-dichlorophenoxy acetic acid (2,4-D) and 1-naphthyl acetic acid (NAA) induced callus in all explants regardless of the concentration. The highest fresh weight of callus (3.92 g) was obtained on MS medium fortified with 1 mg L−1 of both 2,4-D and NAA (DN1) after 7 weeks of culture. DN1 was the best medium for callus multiplication regarding the increase in fresh weight and size of callus. Otherwise, the highest phenolics, flavonoids, and antioxidant activity against DPPH free radicals were of callus on MS fortified with 2 mg L−1 NAA (N2). The cell suspension cultures were cultivated on a liquid N2 medium with different sucrose concentrations of 5–30 g L−1 to observe the possible effects on cells’ multiplication and secondary metabolite production. The highest fresh and viable biomass of 12.01 g was obtained on N2 containing 30 g L−1 sucrose. On the other hand, the cell cultures on N2 medium of 5 and 30 g L−1 sucrose produced phenolics and flavonoids, and revealed antioxidant activity against DPPH and ABTS+ free radicals more than other sucrose concentrations. The presented protocol should be useful in the large-scale production of phenolic and flavonoid compounds from callus and cell cultures of L. schweinfurthii.

Local Infiltrative Analgesia of Murine Femur Fractures In Vivo Does Not Inhibit Fracture Healing.

The opioid epidemic in the United States has forced care providers to seek out alternatives to narcotic analgesics. Physicians involved in trauma care, including orthopaedic trauma surgeons, often have patients requiring significant amounts of these medications, especially in the perioperative period, given the acuity and severity of their injuries. Modalities such as local infiltration of fractures with anesthetic agents during operative treatment may provide some benefit to this population by decreasing postoperative pain and narcotic usage. However, prior data suggest that these agents are chondrotoxic, which may impede secondary fracture healing. The purpose of this study was to investigate the hypothesis that local anesthetics decrease secondary bone healing and callus formation in stabilized murine femur fractures through chondrocyte apoptosis. Male C57BL/6 mice underwent intramedullary stabilization and fracture of bilateral femurs followed by immediate infiltration of the fracture site with local anesthetic agents. Femurs were dissected at 10- and 20-days post-fracture and evaluated by muCT and histological analysis. No significant differences were seen in callus size or mineralization between controls and fractures treated with a local anesthetic. When the callus was analyzed histologically, local anesthetic agents appeared to increase cartilage density. Therefore, infiltration of local anesthetics during operative treatment of fracture as part of a multimodal approach to pain control does not appear to significantly affect callus formation in a preclinical model, although subclinical molecular effects may be present. Local infiltrative analgesia with local anesthetics may be used as an adjunct for perioperative pain control during femur fracture surgery without a significant effect on secondary bone healing.

Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair

Low testosterone level is an independent predictor of osteoporotic fracture in elderly men as well as increased fracture risk in men undergoing androgen deprivation. Androgens and androgen receptor (AR) actions are essential for bone development and homeostasis but their linkage to fracture repair remains unclear. Here we found that AR is highly expressed in the periosteum cells and is co-localized with a mesenchymal progenitor cell marker, paired-related homeobox protein 1 (Prrx1), during bone fracture repair. Mice lacking the AR gene in the periosteum expressing Prrx1-cre (AR-/Y;Prrx1::Cre) but not in the chondrocytes (AR-/Y;Col-2::Cre) exhibits reduced callus size and new bone volume. Gene expression data analysis revealed that the expression of several collagens, integrins and cell adhesion molecules were downregulated in periosteum-derived progenitor cells (PDCs) from AR-/Y;Prrx1::Cre mice. Mechanistically, androgens-AR signaling activates the AR/ARA55/FAK complex and induces the collagen-integrin α2β1 gene expression that is required for promoting the AR-mediated PDCs migration. Using mouse cortical-defect and femoral graft transplantation models, we proved that elimination of AR in periosteum of host mice impairs fracture healing, regardless of AR existence of transplanted donor graft. While testosterone implanted scaffolds failed to complete callus bridging across the fracture gap in AR-/Y;Prrx1::Cre mice, cell-based transplantation using DPCs re-expressing AR could lead to rescue bone repair. In conclusion, targeting androgen/AR axis in the periosteum may provide a novel therapy approach to improve fracture healing.

Mechanically-regulated bone repair.

Fracture healing is a complex, multistep process that is highly sensitive to mechanical signaling. To optimize repair, surgeons prescribe immediate weight-bearing as-tolerated within 24 hours after surgical fixation; however, this recommendation is based on anecdotal evidence and assessment of bulk healing outcomes (e.g., callus size, bone volume, etc.). Given challenges in accurately characterizing the mechanical environment and the ever-changing properties of the regenerate, the principles governing mechanical regulation of repair, including their cell and molecular basis, are not yet well defined. However, the use of mechanobiological rodent models, and their relatively large genetic toolbox, combined with recent advances in imaging approaches and single-cell analyses is improving our understanding of the bone microenvironment in response to loading. This review describes the identification and characterization of distinct cell populations involved in bone healing and highlights the most recent findings on mechanical regulation of bone homeostasis and repair with an emphasis on osteo-angio coupling. A discussion on aging and its impact on bone mechanoresponsiveness emphasizes the need for novel mechanotherapeutics that can re-sensitize skeletal stem and progenitor cells to physical rehabilitation protocols.

In vitro salinity stress mediates grass pea genotypes' (Lathyrus sativus L.) responses

This study was carried out to determine the tolerance of grass pea genotypes to salinity stress at callus and seedling stages under in vitro conditions. The calli and seedlings of six selected tolerant genotypes based on the primary screening in the field were separately exposed to salinity treatments (0, 125 mM) in vitro. Salinity was imposed with NaCl during in vitro culture, and it significantly affected all seedling traits. Genotype of Iran had the lowest seedling dry weight and therefore was more sensitive to salinity stress. According to salinity tolerance indices for seedlings, genotype Greece-III was characterized as high-yield and relatively high?salt-tolerant genotype. Salinity significantly affected callus size, callus RWC, callus RGR, and callogenesis index. Calli fresh and dry weights were not affected by the treatments. For callus dry weight, genotype Greece-III had the highest mean; and the lowest mean belonged to Greece-I. The stress tolerance indices showed that the highest values belonged to genotype Greece-III, which showed high yield and yield stability and so reasonable salinity tolerance. Cluster analysis divided the genotypes into two separate groups. The first cluster consisted of Iran, Greece-II, and Greece-III genotypes, and the second cluster consisted of Bangladesh, Canada, and Greece-I genotypes. Cluster analysis potentially separated the tolerant and sensitive genotypes to salinity in terms of callus dry weight. Grass pea callus and seedlings were able to survive at 125 mM salinity. Salinity did not affect callus dry and fresh weights, but its effect was remarkable on seedling dry and fresh weights (55% less than control). Therefore, calli were reasonably salinity tolerant. The present study suggests that grass pea was reasonably tolerant to salinity and can survive under salinity conditions during the seedling and callus stages.

Novel Fracture Site Targeting Drug Improves Fracture Healing with Pain Relief: Preclinical Evaluation of MAK123

Non-union bone fracture occurs in 5-10% of fracture injuries. Interventions include surgery with local implantation of autograft, allograft, demineralized bone matrix, and/or bone morphogenetic proteins. These types of fracture injuries are also accompanied by acute and chronic pain states. In most instances, opioids are provided to injured patients during and after surgery. With the opioid crisis, identifying new analgesic therapies that could reduce or eliminate opioid use, while also improving bone healing is important. Here we show the ability of a novel compound, MAK123, to both enhance bone healing and reduce pain behavior in a surgically induced femoral fracture mouse model. Briefly, 20 male C57BL/6 mice underwent a surgically induced femoral fracture and then were treated with 0, 2, 6, or 20 mg/kg, 3X/week for the 3 week study duration. Weekly X-rays were used to examine healing progression. Prior to euthanasia, mice underwent behavioral testing to measure evoked pain behaviors. Upon euthanasia, ex vivo µCT imaging and analysis was completed to assess fracture callus size and composition. While all doses of MAK123 tested resulted in improved healing, the 6mg/kg dose resulted in accelerated bone healing and a significant increase in mineralized callus volume (p<0.05). Similarly, while all doses of MAK123 reduced evoked responses to tactile stimulus as demonstrated by increased paw withdrawal thresholds, 6 mg/kg of MAK123 resulted in a more robust and significant improvement (p<0.05). We postulate that optimization of the dosing schedule/concentration could further improve both bone healing and behavioral measures thought to represent pain in rodents. That said, these promising pre-clinical data warrant further evaluation as MAK123 may prove to be a unique tool for orthopaedic surgery usage whereby it could both improve bone healing and reduce clinical pain, improving overall patient outcomes.

The influence of M-CSF on fracture healing in a mouse model

Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice (18-week-old female C57BL/6) which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts.

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model.

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (μCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo μCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).