Callus Formation Research Articles

OBTAINING ASEPTIC CULTURE IN VITRO OF RHODIOLA SEMENOWII L. FROM VARIOUS TYPES OF EXPLANTS

In vitro cultivation of callus cells allows for the stable production of secondary metabolites, being an alternative to the use of medicinal plant biomass from natural populations. Standardization of cultivation protocols and technological processes for the synthesis of secondary metabolites in vitro conditions determines significant interest in the use of tissue culture methods (PTC – Plant Tissue Culture). The aim of the study was to obtain an aseptic culture of Rhodiola semenowii in vitro from various types of explants, as well as the effect of the hormonal composition of the nutrient medium on the induction of callusogenesis in the culture of isolated organs of R. semenowii. As a result of the research, the optimal mode of obtaining R. semenovii aseptic cultures from various explants was determined. It has been shown that the use of 70% EtOH in combination with 0.01% HgCl2 or 20% NaClO makes it possible to obtain aseptic cultures from any type of R. semenovii explants. It has been established that the type of explant is R. semenovii influenced both the frequency of callus formation and the beginning of its formation. The least optimal types of R. semenovii explants are stem and rhizome segments, the frequency of callusogenesis varied from 5.8% to 75.6%. The induction of callusogenesis also depended on the hormonal composition of the nutrient medium: the highest percentage of callus formation in all types of explants was on media containing 0.5mg L−1 thidiazuron (TDZ) + 1.0 mg L−1 1-naphthaleneacetic acid (NAA) and 0.5mg L−12,4D + 2.0 mg L−1 6-Benzylaminopurine (BAP).

Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Endogenous hormone alteration during callus and adventitious root formation through thin cell layer culture system in Phyllanthus amarus

Endogenous hormone alteration during callus and adventitious root formation through thin cell layer culture system in Phyllanthus amarus

Altered post-fracture systemic bone loss in a mouse model of osteocyte dysfunction

Abstract Femur fracture leads to loss of bone at uninjured skeletal sites, which may increase risk of subsequent fracture. Osteocytes, the most abundant bone cells, can directly resorb bone matrix and regulate osteoclast and osteoblast activity, but their role in systemic bone loss after fracture remains poorly understood. In this study we used a transgenic (TG+) mouse model that overexpresses human B-cell lymphoma 2 (BCL-2) in osteoblasts and osteocytes. This causes enhanced osteoblast proliferation, followed by disruption in lacunar-canalicular connectivity and massive osteocyte death by 10 weeks of age. We hypothesized that reduced viable osteocyte density would decrease the magnitude of systemic bone loss after femur fracture, reduce perilacunar remodeling, and alter callus formation. Bone remodeling was assessed using serum biomarkers of bone formation and resorption at 5 days post-fracture. We used micro-computed tomography, high resolution x-ray microscopy, mechanical testing, and Raman spectroscopy to quantify the magnitude of systemic bone loss, as well as changes in osteocyte lacunar volume, bone strength, and bone composition 2 weeks post-fracture. Fracture was associated with a reduction in circulating markers of bone resorption in non-transgenic (TG-) animals. TG+ mice exhibited high bone mass in the limbs, greater cortical elastic modulus and reduced post-yield displacement. After fracture, TG+ mice lost less trabecular bone than TG- mice, but conversely TG+ mice exhibited trends towards a lower yield point and reduced femoral cortical thickness after fracture, though these were not statistically significant. Lacunar density was greater in TG+ mice, but fracture did not alter lacunar volume in TG+ or TG- mice. These findings suggest that osteocytes potentially play a significant role in the post-traumatic systemic response to fracture, though the effects differ between trabecular and cortical bone.

Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants

Fritillaria przewalskii Maxim. an essential medicinal plant in the Fritillaria genus of the Liliaceae family, is widely used in traditional medicine. This study focused on developing a cryopreservation protocol for F. przewalskii, an endangered and endemic species in China. We aimed to determine if ex vitro bulbs from field-grown F. przewalskii plants could serve a source of shoot tips for cryopreservation. Key factors influencing cryopreservation outcomes were studied using a vitrification-based approach. Prior to cryopreservation, cold hardening and sucrose preculture conditions were optimized. The best results were achieved by hardening bulbs at 4℃ for 3-4 months,followed by surface disinfection and isolation of 2-5mm Shoot tips. The tips were then then precultured on a medium with 0.5M sucrose for 3 days. Cryopreservation steps included a 20-minute loading treatment, 60-minute PVS2 dehydration at room temperature, and storage in liquid nitrogen more than one hour. After thawing, this protocol resulted in 93% shoot survival rate, with tips regenerating into small bulbs within 5 weeks without intermediary callus formation. Histological analysis showed that while cooling and rewarming caused severe bud damage, apical meristem cells are the primary survival sites. Although loading and PVS2 treatment induced cell plasmolysis, it was not lethal. Optimal PVS2 treatment enhanced cellular dehydration resistance, promoting cell viability at ultralow temperature. RAPD analysis of regenepercentaged plants showed no genetic variation, confirming strong genetic stability. The direct use of ex vitro shoot tips provides a straightforward and effective cryopreservation method for F. przewalskii, combining convenience, efficiency, and genetic stability.

Accelerating Fracture Healing in Delayed Uniting Fractures: A Clinical Study

Background: Delayed union fractures pose a significant clinical challenge, often requiring enhanced treatment modalities to accelerate healing and improve patient outcomes. Various techniques, including autologous bone grafting and biologic agents like platelet-rich plasma (PRP) and bone morphogenetic proteins (BMPs), have been employed to stimulate bone regeneration. However, the optimal approach remains debated. Objective: This study aimed to compare the efficacy of autologous bone grafting, biologic agents, and combined therapies in promoting fracture healing, reducing pain, improving functional outcomes, and enhancing patient satisfaction in delayed union fractures. Methods: A prospective study was conducted on 80 patients with delayed union fractures. Patients were randomly assigned to one of four groups: standard care (control), autologous bone grafting, biologic agents (PRP and BMPs), or a combination of bone grafting and biologic agents. Radiographic healing was assessed at 6, 12, and 24 weeks. Pain intensity was measured using the Visual Analog Scale (VAS), functional outcomes were assessed using the AAOS lower limb function scale, and patient satisfaction was evaluated using a Likert scale. Results: The combined therapy group exhibited significantly faster radiographic healing, with 80% of patients showing callus formation at 6 weeks and 95% achieving complete union at 24 weeks, compared to 35% in the control group. The combined group also reported the greatest reduction in VAS pain scores, with a mean decrease from 7.8 to 1.5 at 24 weeks (p<0.001). Functional outcomes were significantly improved in the combined group, with a mean AAOS score of 100 at 24 weeks, compared to 70 in the control group (p<0.001). Additionally, 90% of patients in the combined group reported high satisfaction, compared to 40% in the control group. Conclusion: The combination of autologous bone grafting and biologic agents, such as PRP and BMPs, significantly accelerates ..........

Footwear and Foot Health: Unveiling the Role of Proper Shoe Fit in Preventing Podiatric Issues and Enhancing Well-Being

Background: The appropriate choice of footwear is crucial for foot health, yet its impact on different populations and medical conditions remains understudied. This review explores the effect of shoe fit on the prevention of podiatric disorders and overall well-being. Methods: The research included major academic databases such as MEDLINE, Cochrane Central, Scopus, and PEDro, using specific keywords. A scoping review was conducted following the Joanna Briggs Institute methodology, encompassing studies on shoe fit across diverse populations and conditions. Medical databases and grey literature were also included. Results: Five studies were included, covering topics such as footwear advice for women over 50, the effect of shoes in preventing calluses under the metatarsals, the effectiveness of a shoe-related intervention for gout patients, and the impact of custom-fitted shoes on physical activity in children with Down syndrome. Results showed that well-fitting shoes can prevent callus formation, but the efficacy of custom-fitted shoes for increasing physical activity requires further research. Conclusions: The choice of appropriate footwear should not be solely based on aesthetic considerations but rather on the specific needs of each individual. Physicians should consider providing advice on appropriate shoe characteristics as a primary intervention

Effect of the culture liquid of Antarctic yeast <i>Nadsoniella nigra</i> on rooting, growth, and biochemical composition of <i>in vitro</i> grapevine (<i>Vitis vinifera</i> L.), cultivar '<i>Karmrahyut</i>'

Background: Grapevine (Vitis vinifera L.) is a globally significant fruit crop known for its nutritional and health benefits, including its role in preventing chronic diseases such as cancer and cardiovascular disorders. The bioactive compounds in grapes contribute to these benefits. Bioactive content changes due to grapevine variety and environmental conditions necessitate ongoing research. In vitro propagation provides a controlled environment for grapevine cultivation, minimizing disease risk and ensuring consistent quality. This study investigates the potential of Nadsoniella nigra (Nn), an Antarctic yeast, as a natural alternative to synthetic auxins like Indole-3-butyric acid (IBA) in grapevine micro cutting propagation. Objective: To evaluate the effectiveness of the culture liquid of Nn as a natural alternative to auxin-class hormones, using IBA as an example, for promoting rooting in grapevine microcuttings and to assess its impact on chlorophyll content, vitamin C levels, and sugar concentrations. Materials and Methods: In vitro propagation of grapevine (Vitis vinifera L.) cultivar 'Karmarhyt' was performed using half-strength Woody Plant Medium (WPM). Nn culture liquid was prepared at concentrations of 0.5, 1.0, 1.5, and 2.0 ml/L, and IBA was used at equivalent concentrations. Microcuttings were cultured on media supplemented with these treatments, with a control group maintained without growth regulators. Rooting was evaluated after 4 weeks by measuring rooting percentage, number of roots per shoot, and root length. Chlorophyll a (Chl a) and Chlorophyll b (Chl b) levels, vitamin C content, and sugar concentrations were assessed. Statistical analysis was performed using standard error and t-tests. Results: Rooting performance of grapevine microcuttings varied with treatment. Nn culture liquid promoted rooting in a dose-dependent manner, with the highest values observed at 2.0 ml/L (97.0% rooting, 7.2 roots, 13.8 cm root length). IBA treatment resulted in peak rooting at 1.0 mg/L (100% rooting) but decreased at higher concentrations, accompanied by callus formation. Chlorophyll content was higher in Nn culture liquid treated microcuttings, with a maximum total chlorophyll of 2.20 mg/g at 2.0 mg/L, compared to a peak of 2.05 mg/g in IBA-treated microcuttings at 1.0 mg/L. Sugar and vitamin C content increased with Nn culture liquid concentrations, whereas IBA showed a less consistent effect. Conclusion: Nn culture liquid was an effective natural alternative to IBA for promoting rooting in grapevine microcuttings. It improved rooting efficiency, chlorophyll content, and increased sugar and vitamin C levels, while IBA led to growth inhibition and callus formation at higher concentrations. Nn culture liquid demonstrated consistent benefits, suggesting potential for sustainable viticulture. As a result of the conducted research, it was established that the mixture of secondary metabolites contained in the liquid culture medium of Nn possesses high biological activity and can be recommended for use in plant biotechnology in vitro to promote the growth of both underground and above-ground parts. Future research could explore its use in propagating other plant species. Keywords: ´Karmrahyut´, grapevine, culture liquid of Nadsoniella nigra. in vitro propagation, functional foods

Silver Nanoparticles (AgNPs) Act as Nanoelicitors in Melissa officinalis to Enhance the Production of Some Important Phenolic Compounds and Essential Oils

ABSTRACTNanoparticles (NPs) are well‐known biostimulants in plant biotechnology, utilised to enhance the physical properties of plants and exhibit positive effects on them. The important key role is the most suitable type, effective dose and size of NP to be used in plant tissue culture systems. In this study, various concentrations of silver nanoparticles (AgNPs; 0, 25, 50, 75 and 100 μg L−1) were tested as elicitors in callus culture with the aim of enhancing secondary metabolite production in lemon balm (Melissa officinalis L.). According to the results obtained, callus formation rates have shown an increase in all applications compared to the control group. The highest callus formation, weight and diameter were observed in 50 μg L−1 application. In this application, the callus structure was compact and its colour was green. However, the aromatic compounds, neral and geranial increased significantly in 25 μg L−1 application. The maximum increase in phenolic compounds such as caffeic acid, chlorogenic acid, proto‐catechic acid, hesperidin and p‐coumaric acid was observed in the 75 μg L−1 AgNP and the highest increase in rosmarinic acid compound was determined in the 50 μg L−1 application. The study found that AgNP applications are an effective method for increasing the production of secondary metabolites in medicinal and aromatic plants, such as lemon balm, in vitro.

Association of keel bone morphometry with keel bone damage and skeletal quality in the laying hen

ABSTRACT 1. The aim of this work was to identify a heritable keel bone phenotype with a correlation to keel bone damage and/or skeletal bone strength that could be easily measured in the living hen to aid selection to prevent welfare issues. 2. The scoring system used reflected the observed damage, keel bone dimensions and shape compared to traditional measurements of bone quality. 3. Increased keel bone damage was associated with poor humerus and tibia breaking strengths (p < 0.01). Bone damage was associated with higher whole keel density (p < 0.01) due to the effect of callus formation. 4. Keel bone depth and area was moderately heritable at indices of 0.32 and 0.40, respectively. Keel bone depth was genetically correlated with tibia (0.36) and humerus density (0.68) and keel bone area was correlated with humerus density (0.59). Deeper keel bones and those with larger areas had small, negative phenotypic correlations with keel bone damage (−0.07 and −0.11, respectively). The second principal component of keel bone shape represented the caudal section of the keel bone and cranial edge concavity. The third principal component represented the differences in the caudal tip of the keel bone, the concavity of the dorsal edge and convexity of the ventral edge. Heritability estimates were 0.44 and 0.39, respectively. 5. The results suggested that genetics contribute to morphometric traits. Hens with poorer skeletal quality are likely to accumulate more damage. Some of the traits may be a predictor of damage, although mid keel depth or concavity may simply reflect the effect of damage or deviation.

Effects Of 2,4-D, BAP, and Sucrose Concentrations in The Callus Induction of White (Clitoria ternatea var. Albiflora) and Blue Butterfly Pea (Clitoria ternatea)

The blue butterfly pea (Clitoria ternatea) and white butterfly pea (Clitoria ternatea var. Albiflora) belong to the Fabaceae family. Both are locally known as “bunga telang” and native to the Southeast Asian regions. The blue flowered variety is traditionally used to treat headaches, fever, and diabetes and is renowned scientifically for its memory-enhancing properties due to the presence of novel pentacyclic triterpenoids. However, farming of C. ternatea is challenged by inconsistent yields of novel secondary metabolites, especially under changing environmental conditions. Callus and cell suspension cultures, on the other hand, offer an alternative for the consistent production of these metabolites. The current study aims to optimize the treatments of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and sucrose concentrations for friable callus formation from seedling explants. Sterile cotyledon explants of in vitro seedlings from both types of butterfly pea were subjected to half-strength MS medium supplemented with different concentrations and combinations of 2,4-D and BAP, with sucrose at 15 g/L and 30 g/L. The highest friable callus fresh weight from the white butterfly pea explants (0.064 ± 0.010 g) was achieved in treatments of 0.40 mg/L 2,4-D and 0.50 mg/L BAP. In contrast, the highest fresh weight of friable callus for the blue variety (0.025 ± 0.016 g) was induced in 0.25 mg/L of 2,4-D. Both varieties showed the highest friable callus weight in 15 g/L sucrose supplemented with 1.00 mg/L of 2,4-D (0.146 ± 0.032 g) and 0.25 mg/L of 2,4-D (0.245 ± 0.075 g) for the white and blue variety respectively. The morphology of calli for both varieties were yellowish, watery, and sticky. This study provides an essential basis the establishment of cell suspension cultures, as an efficient alternative to harness the secondary metabolites associated with the mammalian neuroprotective properties.

Functional Outcome of Extra-Articular Distal Tibia Fracture Treated with Tibia Interlocking Nail: A Case Series

Background: Intramedullary interlocking nailing has emerged as one of the suitable management options for distal tibia fractures as it allows mechanical compression on weight-bearing, which stimulates bone healing. We studied 40 cases of extra-articular distal tibia fracture treated by intramedullary nailing. Methods: We retrospectively studied 40 patients of both sexes above the age of 18 years with closed fractures and grade 1, 2, and 3A compound fractures treated at SVP Hospital, Ahmedabad, India. Clinically, the fracture was considered to be united when the patient was completely pain-free. Patients were followed up at monthly intervals till union. At each follow-up, patients were assessed both clinically and radiographically, and details were recorded based on proforma. Results: The evaluation of the result was done using American Orthopedic Foot and Ankle Society (AOFAS) criteria. Thirty-three patients had excellent functional results, five had good functional results, and Two had fair functional results. In this study, 38 (95%) patients had radiological union within 24 weeks. Two patients had union after 28 weeks. Conclusion: Intramedullary nailing is a minimally invasive procedure that preserves the soft tissue and the fracture hematoma. It maintains the length, alignment, and rotation and allows micromotion at the fracture site on weight-bearing, which stimulates callus formation. It has a good functional outcome with gratifying results when used in extra-articular distal tibia fractures.

Analyzing the temporal response mechanisms of the vascular bundles formation of grafted cucumber to different light intensity modes: A joint transcriptomic and metabolomic approach

In plant grafting, the reconnection of vascular bundles between the rootstock and scion not only establishes functional xylem and phloem connections, but also signifies the completion of graft healing. In our previous studies focused on early-stage morphology and physiology of grafted seedlings, we have demonstrated that utilizing light intensities of 50-100-150 μmol/(m2·s) during days 1-3, 4-6, and 7-9 optimizes cucumber/pumpkin graft healing. To further explore the underlying mechanisms, we investigate light intensity regimes: 50-100-100 μmol/(m2·s) and 50-100-150 μmol/(m2·s) during the same time frames (days 1-3, 4-6, and 7-9 after grafting), denoted as CK and T treatments. Initially, we observe histological characteristics of vascular bundle formation under these conditions, followed by the analyses of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-Px) enzyme activities, ascorbic acid (AsA) content in the antioxidant system, and the sequencing of transcriptome and metabolome. It is found that grafted seedlings under the T treatment achieve faster vascular bundle reconnection and shorter healing process compared to those under the CK treatment. Enzyme activities (POD, SOD, GSH-Px) increase over time under both light regimes, while CAT activity and AsA content decrease. Notably, by the 9th day, the T treatment (T9d) exhibits higher SOD, POD and CAT activities than the CK treatment (CK9d). Our findings indicate that increasing light intensity during the days 7-9 after grafting can mitigate oxidative damage at the grafting site and benefit vascular bundle reconnection. Transcriptome and metabolome analyses show the up-regulations of Cinnamaldehyde in the phenylpropanoid biosynthesis pathway, the PHGDH gene and L-Homocysteine in the cysteine and methionine metabolism pathway under T treatment in the days 7-9 after grafting. These enhance the antioxidant enzyme activities and reduce the oxidative damage to the grafting site. Additionally, GAE-related genes, and metabolites D-Xylose and Undecaprenyl phosphatealpha-L-Ara4FN in the amino sugar and nucleotide sugar metabolism pathways are also up-regulated under T treatment. The ethylene responsive factor (ERF) transcription factor CsaRAP2.3 positively regulates Undecaprenyl phosphatealpha-L-Ara4FN expression, promoting cell wall synthesis and therefore accelerating vascular bundle reconnection. In summary, during the vascular bundle formation period of cucumber graft healing, both light intensity modes coordinate regulation of phenylpropanoid biosynthesis, cysteine and methionine metabolism, amino sugar and nucleoside sugar metabolism, and ERF transcription factors, promoting graft healing from both stress resistance and cell wall synthesis. Furthermore, building upon the light intensity applied during the callus formation phase, increasing the light intensity during the vascular bundle formation period can significantly enhance cell wall synthesis, thus expediting the graft healing process. Nonetheless, the generalizability of these research findings to other grafting techniques and species requires further investigation.

Innovative approaches to cage reconstructions in orthopedic limb surgery: Advances and insights with two cases

BackgroundLimb tumors, though rare, pose significant challenges to surgical management due to their impact on limb function and quality of life. Effective treatment requires precise tumor resection alongside advanced reconstructive techniques to restore limb function, particularly in cases of compromised bone healing. Titanium cages, traditionally used in spinal surgeries, have recently gained attention for their potential in limb reconstruction within orthopedic oncology, offering a novel approach to managing complex bone defects. Case PresentationThis retrospective case series presents two patients treated with titanium cages for limb reconstruction following tumor resection.Case 1: A 53-year-old female with a solitary plasmacytoma of the femur experienced a pathological fracture and nonunion after initial treatment. A titanium cage was employed to bridge the resected bone segment, leading to successful callus formation at the six-month follow-up and restored limb function.Case 2: A 31-year-old female with a giant cell tumor and hemangioma of the foot underwent en bloc resection followed by reconstruction using a vertebral cage and iliac corticospongious graft. Six months postoperatively, the patient had resumed full weight-bearing with no complications. ConclusionTitanium cages offer a promising solution for limb reconstruction in patients with tumors, particularly for large bone defects where traditional methods may be inadequate. This case series demonstrates successful outcomes in limb preservation and functional recovery using titanium cages. Their use in the adult population shows potential for broader application in orthopedic oncology, warranting further clinical investigation.

Downregulation of Krüppel-like factor 15 expression delays endochondral bone ossification during fracture healing

ObjectiveThe role of Krüppel-like zinc finger transcription factor 15 (KLF15) in endochondral ossification during fracture healing remains unexplored. In this study, we aimed to elucidate the impact of KLF15 in a mouse model of tibial transverse fracture. MethodsWe created tamoxifen-inducible, cartilage-specific KLF15 knockout mice (KLF15 KO). KLF15 fl/fl Col2-CreERT mice from the same litters as the KLF15 KO mice, but not treated with 4-hydroxytamoxifen, were used as controls (CT). At 10 weeks, male KLF15 KO and CT mice underwent tibial fracture followed by intramedullary nailing. Both groups were administered tamoxifen at days 0, 3, and 7 after surgery. The tibiae were harvested on post-surgery days 7, 10, and 14 for radiological assessment using micro-computed tomography. Histological staining (Safranin-O) and immunohistochemistry for KLF15, SOX9, Indian hedgehog (IHH), RUNX2, and Osterix were performed. Additionally, cartilage from mouse fetus was cultured for qRT-PCR and western blot analyses of KLF15, SOX9, IHH, Col2, RUNX2, Osterix, TGF-β, SMAD3, and phosphor-SMAD3. ResultsThe radiological assessment revealed that immature callus formation was delayed in KLF15 KO, compared with that in CT, peaking on day 14 compared with that on day 10 in CT. KLF15 KO mice exhibited delayed fracture healing and reduced Safranin-O staining at days 7 and 10 post-surgery. The ratio of cells positive for KLF15 and SOX9 was significantly lower in KLF15 KO than in CT, whereas the ratios for IHH, RUNX2, and Osterix showed no significant difference. RT-PCR revealed reduced expression of KLF15, SOX9, and COL2, with no significant changes in IHH, Osterix, RUNX2, TGF-β, and SMAD3. Western blot analysis indicated decreased SMAD3 phosphorylation in KLF15 KO mice. ConclusionKLF15 regulates SOX9 via the TGF-β-SMAD3-SOX9 pathway, independent of IHH, in endochondral ossification. The KLF15 deficiency decreases SOX9 expression through reduced SMAD3 phosphorylation, subsequently delaying fracture healing.

A Cyclin Gene OsCYCB1;5 Regulates Seed Callus Induction in Rice Revealed by Genome Wide Association Study

Plant tissue culture is extensively employed in plant functional genomics research and crop genetic improvement breeding. The callus induction ability is critical for utilizing Agrobacterium-mediated genetic transformation. In this study, we conducted a genome-wide association study (GWAS) utilizing 368 rice accessions to identify traits associated with callus induction rate (CIR), resulting in the identification of a total of 104 significant SNP loci. Integrated with gene function annotation and transcriptome analysis, 13 high-confidence candidate genes involved in auxin-related, CYC cyclins, and histone H3K9-specific methyltransferase were identified in significant loci. Furthermore, we also verified a candidate gene, Os05g0493500 (OsCycB1;5), and employed the CRISPR/Cas9 system to generate OsCycB1;5 knockout mutants in rice (Oryza sativa L.). The OscycB1;5 mutant displays significantly reduced callus induction and proliferation capacity, this result indicating OsCycB1;5 is required for the callus formation in rice. Overall, this study provides several reliable loci and high-confidence candidate genes that may significantly affect callus formation in rice. This information will offer valuable insights into the mechanisms underlying callus formation not only in rice but also in other plants.

Biomechanical Evaluation of Hydroxyapatite/poly-l-lactide Fixation in Mandibular Body Reconstruction with Fibula Free Flap: A Finite Element Analysis Incorporating Material Properties and Masticatory Function Evaluation.

In reconstructive surgery following partial mandibulectomy, the biomechanical integrity of the fibula free flap applied to the remaining mandibular region directly influences the prognosis of the surgery. The purpose of this study is to evaluate the biomechanical integrity of two fixation materials [titanium (Ti) and hydroxyapatite/poly-L-lactide (HA-PLLA)]. In this study, we simulated the mechanical properties of miniplate and screw fixations in two different systems by finite element analysis. A three-dimensional mandibular model was constructed and a fibula free flap and reconstruction surface were designed. The anterior and posterior end of the free flap was positioned with two miniplates and two additional miniplates were applied to the angled area of the fibula. The masticatory loading was applied considering seven principal muscles. The peak von Mises stress (PVMS) distribution, size of fixation deformation, principal stresses on bones, and gap opening size were measured to evaluate the material properties of the fixation. In the evaluation of properties, superior results were observed with both fixation methods immediately after surgery. However, after the formation of callus between bone segments at 2 months, the performance of Ti fixation decreased over time and the differences between the two fixations became minimal by 6 months after surgery. The result of the study implies the positive clinical potential of the HA-PLLA fixation system applied in fibula free flap reconstruction.

Cigarette Smoke Exposure Impairs Fracture Healing in a Rat Model: Preferential Impairment of Endochondral over Membranous Healing.

Cigarette smoking affects fracture repair, leading to delayed healing or nonunion. We sought to investigate if cigarette smoke differentially affects intramembranous and endochondral ossification in healing fractures, focusing on whether endochondral ossification is particularly impaired. This study utilized a bilateral femur fracture model in Sprague Dawley rats to examine the impact of cigarette smoke exposure on healing of femur fractures, treated with either a custom-locked intramedullary nail or compression plating to induce endochondral and membranous ossification, respectively. Animals were exposed to tobacco smoke 30 days before and after surgery, with evaluations including radiographs, histomorphometry, and microCT at 10 days, 1, 3, and 6-months post-operation, and biomechanical testing at 3, 6 months. Sixty-eight animals were randomized to control or exposure groups (two died perioperatively), and 89% of the femora achieved union when harvested at 3 and 6 months. Smoke exposure delayed cartilaginous callus formation and bone maturation in nailed fractures compared to plated fractures and controls in same animals. Plated fractures in exposed animals exhibited little cartilage callus and healed like control animals. At 3 months, plated fractures were stiffer and stronger than nailed fractures in both groups, but these differences vanished by 6 months. Plated fractures healed more rapidly and more completely than nailed fractures under both control and smoke-exposed conditions. Using compression plating instead of IM nailing for closed long bone fractures may lead to better outcomes in patients who smoke compared to current results with nailing.

Bone and periosteum protein analysis via tandem mass tag quantitative proteomics in pediatric patients with osteomyelitis.

Bone healing is crucial in managing osteomyelitis after fracture fixation. Understanding the mechanism of extensive callus formation in pediatric osteomyelitis is highly important. This study aims to analyze bone and periosteum samples from pediatric patients to elucidate the essential processes involved in callus formation during osteomyelitis. The study included eight patients from our hospital: four with positive microbial culture who underwent osteomyelitis debridement and four who had osteotomy surgery as contral. We used tandem mass tag quantitative proteomics to investigate proteomic changes in bone and periosteum tissues obtained from these patients. Differential expression proteins were analyzed for their pathways through Gene Ontology (GO) annotation, GO enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction networks. A total of 4737 proteins were successfully identified. About 2224 differentially expressed proteins were detected in the bone tissues group and periosteum tissues group. Among the differentially expressed proteins, 10 protein genes in the bone group were associated with inflammation and osteogenesis, while in the periosteum group were nine. Cytochrome b-245, beta polypeptide (CYBB), nicotinamide phosphoribosyltransferase (NAMPT), tissue inhibitor of metalloproteinases 1 (TIMP-1), Raf-1 proto-oncogene, serine/threonine kinase (RAF-1), RELA proto-oncogene, NF-KB subunit (RELA), and sphingomyelin synthase 2 (SGMS2) may play an important role in callus formation in patients with osteomyelitis. This study provides novel clues for understanding callus formation in pediatric patients with osteomyelitis.

Growth Differentiation Factor 5-Induced Mesenchymal Stromal Cells Enhance Tendon Healing.

Mesenchymal stromal cells (MSCs) have immense potential for use in musculoskeletal tissue regeneration; however, there is still a paucity of evidence on the effect of tenogenic MSCs (TMSCs) in tendon healing invivo. This study aimed to determine the effects of growth differentiation factor 5 (GDF5)-induced rabbit MSCs (rbMSCs) on infraspinatus tendon healing in a New Zealand white rabbit model. In this study, bone marrow-derived rbMSCs were isolated, and 100 ng/mL GDF5 was used to induce tenogenic differentiation in rbMSC. The effects of GDF5 on rbMSC in vitro were assessed by total collagen assay, gene expression analysis, and immunofluorescence staining of tenogenic markers; native tenocytes isolated from rabbit tendon were used as a positive control. In invivo, a window defect was created on the infraspinatus tendons bilaterally. After 3 weeks, the rabbits (n = 18) were randomly divided into six groups and repaired with various interventions: (1) surgical suture; (2) fibrin glue (FG); (3) suture and FG; (4) suture, FG, and rabbit tenocytes (rbTenocyte); (5) suture, FG, and rbMSCs, and (6) suture, FG, and TMSC. All animals were euthanized at 6 weeks postoperatively. The in vitro GDF5-induced rbMSCs (or TMSC) showed increased total collagen expression, augmented scleraxis (SCX), and type-I collagen (COL1A1) mRNA gene expression levels. Immunofluorescence showed similar expression in GDF5-induced rbMSC to that of rbTenocyte. In vivo histological analysis showed progressive tendon healing in the TMSC-treated group; cells with elongated nuclei aligned parallel to the collagen fibers, and the collagen fibers were in a more organized orientation, along with macroscopic evidence of tendon callus formation. Significant differences were observed in the cell-treated groups compared with the non-cell-treated groups. Histological scoring showed a significantly enhanced tendon healing in the TMSC- and rbMSC-treated groups compared with the rbTenocyte group. The SCX mRNA expression levels, at 6 weeks following repair, were significantly upregulated in the TMSC group. Immunofluorescence showed COL-1 bundles aligned in parallel orientation; this was further confirmed in atomic force microscopy imaging. SCX, TNC, and TNMD were detected in the TMSC group. In conclusion, GDF5 induces tenogenic differentiation in rbMSCs, and TMSC enhances tendon healing invivo compared with conventional suture repair. Impact Statement Tendon tears and degeneration are debilitating clinical conditions. To date, the suture method is the only gold standard for repairing tendons. Mesenchymal stromal cells (MSCs) have been suggested for many years for their potential in tissue regeneration, especially in tendon-degenerative conditions. Growth differentiation factor 5 (GDF5) has been reported to induce human MSC into a tenogenic lineage (or TMSC), hence a potential cell source for tendon regeneration. This study reported on the potential of rabbit MSC to differentiate into TMSC via GDF5 induction and the potential of TMSC in tendon healing in a New Zealand white rabbit infraspinatus tendon model fulfilled with the 3R principle (reduce, reuse, and replace).