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1.
Article in Chinese | WPRIM | ID: wpr-1009114

ABSTRACT

OBJECTIVE@#To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits.@*METHODS@#TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties.@*RESULTS@#CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05).@*CONCLUSION@#TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.


Subject(s)
Rabbits , Animals , Rotator Cuff/surgery , Chitosan , Hydrogels , Rotator Cuff Injuries/surgery , Wound Healing , Tendons/surgery , Collagen , Stem Cells , Biomechanical Phenomena
2.
Chin. med. j ; Chin. med. j;(24): 329-337, 2024.
Article in English | WPRIM | ID: wpr-1007634

ABSTRACT

BACKGROUND@#Pathological scars are a disorder that can lead to various cosmetic, psychological, and functional problems, and no effective assessment methods are currently available. Assessment and treatment of pathological scars are based on cutaneous manifestations. A two-photon microscope (TPM) with the potential for real-time non-invasive assessment may help determine the under-surface pathophysiological conditions in vivo . This study used a portable handheld TPM to image epidermal cells and dermal collagen structures in pathological scars and normal skin in vivo to evaluate the effectiveness of treatment in scar patients.@*METHODS@#Fifteen patients with pathological scars and three healthy controls were recruited. Imaging was performed using a portable handheld TPM. Five indexes were extracted from two dimensional (2D) and three dimensional (3D) perspectives, including collagen depth, dermo-epidermal junction (DEJ) contour ratio, thickness, orientation, and occupation (proportion of collagen fibers in the field of view) of collagen. Two depth-dependent indexes were computed through the 3D second harmonic generation image and three morphology-related indexes from the 2D images. We assessed index differences between scar and normal skin and changes before and after treatment.@*RESULTS@#Pathological scars and normal skin differed markedly regarding the epidermal morphological structure and the spectral characteristics of collagen fibers. Five indexes were employed to distinguish between normal skin and scar tissue. Statistically significant differences were found in average depth ( t = 9.917, P <0.001), thickness ( t = 4.037, P <0.001), occupation ( t = 2.169, P <0.050), orientation of collagen ( t = 3.669, P <0.001), and the DEJ contour ratio ( t = 5.105, P <0.001).@*CONCLUSIONS@#Use of portable handheld TPM can distinguish collagen from skin tissues; thus, it is more suitable for scar imaging than reflectance confocal microscopy. Thus, a TPM may be an auxiliary tool for scar treatment selection and assessing treatment efficacy.


Subject(s)
Humans , Cicatrix/diagnostic imaging , Skin/pathology , Collagen , Imaging, Three-Dimensional/methods
3.
São Paulo; s.n; s.n; 2024. 153 p tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1563343

ABSTRACT

A reconstrução de modelos avançados de pele tridimensional in vitro, que corresponda de forma mais fidedigna ao complexo microambiente da pele humana, depende da utilização de inovações tecnológicas e da adição de novos tipos celulares representativos da pele humana. Desta maneira, estes miméticos fornecem uma plataforma de alta relevância para estudos de fisiopatologia da pele, além de propiciar um sistema para a avaliação da segurança e eficácia de cosméticos e medicamentos alternativo ao uso de animais. Dessa maneira, o Capítulo I compara a performance de uma epiderme reconstruída humana (RHE) bioimpressa com a manual utilizando o teste in vitro de irritação cutânea descrito no guia OCDE número 439. Nossos resultados demonstram que ambos os modelos de pele exibiram morfologia estratificada e a função barreira epidérmica equivalente aos modelos validados. Nos testes de irritação in vitro, ambos modelos distinguiram corretamente as substâncias de referência, classificadas entre irritantes ou não-irritantes de acordo com o limiar de viabilidade de 50%. Esse resultado indica que a bioimpressora poderia ser de grande utilidade para a automação da reconstrução de modelos epidérmicos. O tecido hipodérmico possui importante papel na homeostase da pele humana. O Capítulo II aborda a reconstrução de uma pele tricamada, contendo a camada hipodérmica, além da epiderme e derme. Usando esferoides de adipócitos diferenciados in vitro, um modelo de pele tricamada em matriz de colágeno foi construído. Ao comparar este com a pele bicamada obtivemos maior expressão de loricrina e involucrina no modelo tricamada, indicando um potencial para maior função barreira, além de maior expressão de PPAR-γ. Testes de função barreira através da resistividade elétrica não demonstraram diferenças entre os modelos, mas a aplicação de SDS a 5 mg/ml por 18 horas induziu o aumento da viabilidade na pele tricamada. Além disso, após a aplicação de SDS a 2,5% para induzir uma irritação aguda, seguida de recuperação por 42h, obtivemos maior viabilidade na pele tricamada, indicando melhor recuperação pós-lesão irritativa induzida. A pele tricamada é promissora para estudos do metabolismo da pele humana e recuperação de lesões. A dermatite atópica (DA) é uma doença eczematosa de pele caracterizada por inflamação do tipo Th2 e alteração da barreira epidérmica. IL-13 e IL-4 são centrais no comprometimento da barreira epidérmica na DA. Entre os receptores de IL-13 em queratinócitos, o receptor IL-13Rα2, tem um papel controverso na alteração da barreira cutânea. O objetivo do Capítulo III foi estudar a deleção da expressão de IL-13Rα2 em RHE, que foram expostas a IL-4 e IL-13, e avaliadas conforme a expressão dos receptores e de proteínas alteradas na DA. As epidermes com knockout em IL-13Rα2 apresentaram redução da expressão de NELL2 (p<0,0021), tipicamente aumentadas na DA. Além disso, houve redução da expressão do receptor do IL-2Rγ. Assim, um possível papel de exacerbação da DA do receptor IL-13Rα2 deve ser estudado mais extensamente para ser caracterizado


The reconstruction of advanced three-dimensional in vitro skin models, which more reliably correspond to the complex microenvironment of human skin, depends on the use of technological innovations and the addition of new cell types representative of human skin.In this way, these mimetics provide a highly relevant platform for studies of skin pathophysiology, in addition to providing a system for evaluating the safety and efficacy of cosmetics and medicines alternative to animal use. In this way, Chapter I compares the performance of a bioprinted human reconstructed epidermis (RHE) with a manual one using the in vitro skin irritation test described in OECD guide number 439. Our results demonstrate that both skin models exhibited stratified morphology and the epidermal barrier function equivalent to validated models. In in vitro irritation tests, both models correctly distinguished the reference substances, classified as irritating or non-irritating according to the viability threshold of 50%. This result indicates that the bioprinter could be of great use for automating the reconstruction of epidermal models Hypodermic tissue plays an important role in the homeostasis of human skin. Chapter II addresses the reconstruction of a three-layer skin, containing the hypodermic layer, in addition to the epidermis and dermis. Using in vitro differentiated adipocyte spheroids, a trilayer skin model in collagen matrix was constructed. When comparing this with bilayer skin, we obtained greater expression of loricrin and involucrin in the trilayer model, indicating a potential for greater barrier function, in addition to greater expression of PPAR-γ . Barrier function tests using electrical resistivity did not demonstrate differences between the models, but the application of SDS at 5 mg/ml for 18 hours induced an increase in viability in the three-layer skin. Furthermore, after applying 2.5% SDS to induce acute irritation, followed by recovery for 42 hours, we obtained greater viability in the three-layer skin, indicating better recovery after induced irritant injury. Trilayer skin holds promise for studies of human skin metabolism and injury recovery. Atopic dermatitis (AD) is an eczematous skin disease characterized by Th2-type inflammation and alteration of the epidermal barrier. IL-13 and IL-4 are central to the impairment of the epidermal barrier in AD. Among the IL-13 receptors on keratinocytes, the IL-13Rα2 receptor has a controversial role in altering the skin barrier. The objective of Chapter III was to study the deletion of IL-13Rα2 expression in RHE, which were exposed to IL-4 and IL-13, and evaluated according to the expression of receptors and proteins altered in AD. Epidermis with IL-13Rα2 knockout showed reduced NELL2 expression (p<0.0021), typically increased in AD. Furthermore, there was a reduction in the expression of the IL-2Rγ receptor. Therefore, a possible AD exacerbation role of the IL-13Rα2 receptor should be studied more extensively to be characterized


Subject(s)
Skin/physiopathology , Dermatitis, Atopic/pathology , Wounds and Injuries/physiopathology , In Vitro Techniques/methods , Pharmaceutical Preparations/analysis , Collagen/agonists , Cosmetics/classification , Epidermis/physiopathology , Inflammation/classification
4.
Acta cir. bras ; Acta Cir. Bras. (Online);39: e393724, 2024. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1563646

ABSTRACT

Purpose: To evaluate collagen fibers during the bone repair process in critical defects created in the tibias of rats, treated with zoledronic acid (AZ) associated with low-level laser therapy (LLLT). Methods: Ten rats were distributed according to treatment: group 1) saline solution; group 2) LLLT; group 3) AZ; group 4) AZ and LLLT. AZ was administered at the dose of 0.035 mg/kg at fortnightly intervals over eight weeks. Next, 2-mm bone defects were created in the tibias of all animals. The bone defects in groups 2 and 4 were irradiated LLLT in the immediate postoperative period. After periods 14 and 28 of application, the animals were euthanized, and birefringence analysis was performed. Results: Approximately 90% of the total area was occupied by collagen fibers within the red color spectrum, this area being statistically larger in relation to the area occupied by collagen fibers within the green and yellow spectrum, in the four groups. Over the 14-day period, there was no statistically significant difference between the groups. In the 28-day period, group 2 (14.02 ± 15.9%) was superior in quantifying green birefringent fibers compared to group 1 (3.06 ± 3.24%), with p = 0.009. Conclusions: LLLT associated with ZA is effective in stimulating the neoformation of collagen fibers. The LLLT group without the association with ZA showed a greater amount of immature and less organized matrix over a period of 28 days.


Subject(s)
Animals , Rats , Bone and Bones , Collagen , Low-Level Light Therapy , Zoledronic Acid/therapeutic use
5.
Braz. j. oral sci ; 23: e244006, 2024. ilus
Article in English | LILACS, BBO | ID: biblio-1553400

ABSTRACT

Aim: The aim of this study was to investigate the impact of pretreatment with ethanolic solutions of caffeic acid phenethyl ester (CAPE) at varying concentrations on the dentin collagen matrix, specifically focusing on its biomodification potential. This was assessed through evaluations of the modulus of elasticity and changes in mass. Methods: Seventy dentin collagen matrices (demineralized sticks) were prepared to receive treatments with ethanolic solutions of CAPE at concentrations of 0.05%, 0.1%, 0.5%, or 2.5%, or with control treatment solutions (distilled water or ethanol) for one hour. The dentin matrices were evaluated for modulus of elasticity and mass before (baseline), immediately after treatment (immediately), and after storage in Simulated Body Fluid (SBF) for time intervals of 1 and 3 months. Results: Generalized linear models for repeated measures over time indicated no significant differences between groups (p=0.7530) or between different time points (p=0.4780) in terms of the modulus of elasticity. Regarding mass variation, no differences were observed in the time interval between 1 month and the immediate time (p=0.0935). However, at the 3-month mark compared to the immediate time, the 0.1% CAPE group exhibited less mass loss compared to the water group (p=0.0134). Conclusion: This study concludes that various concentrations of CAPE in an ethanolic solution did not affect the modulus of elasticity of dentin, suggesting that CAPE lacks biomodifying potential in this context. However, it was observed that 0.1% CAPE positively influenced the variation in mass over different evaluation time intervals


Subject(s)
Caffeic Acids , Collagen , Dentin , Ethanol , Linear Models
6.
Braz. j. biol ; 84: e253616, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1355880

ABSTRACT

Abstract This study evaluated the effect of the volatile oil of Alpinia zerumbet (VOAz) on caveolin-1 gene expression and muscular fibrosis. The rats were immobilized to induce fibrosis of the gastrocnemius muscle, and they were treated with VOAz. Collagen quality was assessed by histology and the expression of the caveolin-1 (CAV-1) gene was evaluated using qPCR. Histomorphological analysis indicated a significant reduction in the perimeter, width, and intensity of collagen in the treated groups, thus showing that the oil was effective in regulating the quality of collagen at the three concentrations. The results of expression levels suggested a decrease in the lesioned group and in two treatment groups (0.0115 µg/g and 0.009 µg/g). However, with the lowest concentration (0.0065 µg/g), no significant difference was observed, with levels similar to those found in healthy tissue. Therefore, the results showed that VOAz has the potential to be a non-invasive and low-cost alternative to aid in the treatment of muscular fibrosis.


Resumo Este estudo avaliou o efeito do óleo volátil de Alpinia zerumbet (OVAz) na expressão do gene da caveolina-1 e na fibrose muscular. Os ratos foram imobilizados para induzir a fibrose do músculo gastrocnêmio, e foram tratados com OVAz. A qualidade do colágeno foi avaliada com histologia e à expressão do gene caveolina-1 (CAV-1) foi avaliada usando qPCR. A análise histomorfológica indicou uma redução significativa no perímetro, largura e intensidade do colágeno nos grupos tratados. Os resultados dos níveis de expressão sugeriram diminuição nos grupos de lesão e em dois grupos de tratamento (0,0115 µg/g e 0,009 µg/g). No entanto, com a menor concentração (0,0065 µg/g), não foi observada diferença significativa, apresentando níveis semelhantes aos encontrados em tecido saudável. O uso do OVAz foi eficaz para reverter as alterações do colágeno causadas pela fibrose, e sua menor concentração apresentou uma possível tendência de aumento na expressão do CAV-1. Portanto, os resultados mostraram que o OVAz tem potencial para ser uma alternativa não invasiva e de baixo custo para auxiliar no tratamento da fibrose muscular.


Subject(s)
Animals , Rats , Oils, Volatile/pharmacology , Collagen/metabolism , Alpinia/chemistry , Caveolin 1/metabolism , Muscles/drug effects , Fibrosis , Plant Oils/pharmacology , Brazil , Rats, Wistar , Disease Models, Animal , Muscles/pathology
7.
Acta cir. bras ; Acta Cir. Bras. (Online);39: e399424, 2024. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1581520

ABSTRACT

Purpose: To evaluate the association of collagen gel with F18 bioactive glass (BG) in the healing of non-contaminated cutaneous wounds induced in healthy Wistar rats. Methods: One hundred twelve adult and healthy Wistar rats were randomly divided into four groups (n = 28): saline solution (0.9%); healing ointment based on allantoin and zinc oxide; collagen gel; and association of F18 BG powder and collagen gel. All the rats underwent the creation of a 3-cm diameter wound in their dorsal region. Macroscopic, thermographic, and tensiometric evaluations of the wound were performed. Results: The presence of granulation tissue varied significantly in and between the groups. The surface temperature assessed through thermography of wounds treated with saline solution (0.9%) increased significantly over time and between the groups. No difference was identified regarding tensiometry. Conclusions: Collagen gel associated with F18 BG induced beneficial healing effects on non-contaminated cutaneous wounds in Wistar rats, which included the induction of increased blood perfusion as assessed through thermography.


Subject(s)
Wound Healing , Wounds and Injuries , Biocompatible Materials , Collagen
8.
J. coloproctol. (Rio J., Impr.) ; 43(2): 68-74, Apr.-June 2023. tab
Article in English | LILACS | ID: biblio-1514425

ABSTRACT

Introduction: The management of complex anal fistulae remains a topical surgical problem. The choice and success of surgical management are based on the balance between healing and continence. Although porcine dermal collagen (Permacol Collagen Paste [PCP]- Covidien plc, Gosport, Hampshire, UK) represents a new generation of non-solid biomaterials, its results in anal fistulae are mixed. Methods: A multicenter observational retrospective analysis of consecutive patients with cryptoglandular anal fistula treated in four colorectal surgery units was performed between 2015 and 2020. Clinical cure of the fistula was the main outcome measure. Adverse events and alterations in anal continence were secondary outcomes. Results: The study included 119 patients (87 males, 71.1%), with a mean age of 53 years (IR 44-65). Most patients had complex (80.6%) and recurrent (91.6%) fistulae. With the first PCP treatment, the overall cure rate was 41.2% (49 patients) and 45.4% with the second treatment (5 out of 17 patients). The mean follow-up period was 17 months (IR 5-25). Healing was not affected by the location and type of fistula, the existence or not of a cavity, the number of tracts, or the administration of prophylactic antibiotics. After the PCP treatment, no patient in the series had worsening of continence. Morbidity affected 22.7% of the patients (27), with postoperative abscesses being the most frequent adverse event. There were no statistical differences between the four hospitals studied. Conclusions: Permacol collagen paste is a safe and easily reproducible therapy for complicated anal fistulae that has moderate efficacy. The overall success rate is slightly over 40%, with no detriment to fecal continence. (AU)


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Collagen/therapeutic use , Rectal Fistula/therapy , Recurrence , Swine , Health Profile , Cohort Studies , Treatment Outcome
9.
Int. j. odontostomatol. (Print) ; 17(1): 101-106, mar. 2023. ilus, graf
Article in English | LILACS | ID: biblio-1430547

ABSTRACT

Inflammatory fibrous hyperplasia (IFH) is a common reactive lesion in dental prostheses users that may be associated with chondroid metaplasia (CM). Metaplasia is an adaptive cellular process that may be caused by trauma. We reported here five cases of IFH associated with CM and analyzed morphologically the deposition of collagen in these lesions. Patients had a mean age of 58.8 years-old and were ill-fitting dental prostheses users. They presented nodular lesions located in the anterior maxilla. Microscopically, it was observed hyperplastic fibrous connective tissue with chronic inflammatory infiltrate and hyaline cartilage. No morphological differences were observed in collagen deposition under light microscopy, but quantitative analysis revealed a significantly higher collagen deposition at the connective tissue near CM (p = 0.015). IFH associated with CM affects ill-fitting dental prostheses users. The presence of CM is not significant to the lesion prognosis. However, its formation and the higher collagen deposition near it reinforces the IFH reactive origin.


La hiperplasia fibrosa inflamatoria (HFI) es una lesión reactiva común en los usuarios de prótesis dentales que puede estar asociada con la metaplasia cartilaginosa (MC). La metaplasia es un proceso celular adaptativo que puede ser causado por un trauma. El presente informe analizó cinco casos de HFI asociados a MC y se analizaron morfológicamente la deposición de colágeno en estas lesiones. Los pacientes tenían una edad media de 58,8 años y eran usuarios de prótesis dentales mal adaptadas. Se observaron lesiones nodulares localizadas en el la parte anterior del maxilar Microscópicamente se observó tejido conectivo fibroso hiperplásico con infiltrado inflamatorio crónico y cartílago hialino. No se observaron diferencias morfológicas en la deposición de colágeno bajo microscopía óptica, pero el análisis cuantitativo reveló una deposición de colágeno significativamente mayor en el tejido conectivo cerca de MC (p = 0,015). La HFI asociada con la MC afecta a los usuarios de prótesis dentales mal adaptadas. La presencia de MC no es significativa para el pronóstico de la lesión. Sin embargo, su formación y la mayor deposición de colágeno cerca de MC refuerza el origen reactivo de HFI.


Subject(s)
Humans , Female , Middle Aged , Aged , Mouth/pathology , Mouth Mucosa/pathology , Fibrosis , Radiography, Dental , Cartilage/pathology , Collagen , Hyperplasia/pathology
10.
Rev. Bras. Ortop. (Online) ; 58(1): 72-78, Jan.-Feb. 2023. tab, graf
Article in English | LILACS | ID: biblio-1441335

ABSTRACT

Abstract Objective To evaluate the effects of hydrolyzed collagen and collagen peptide in the treatment of superficial chondral lesions in rats. Method This research employed 18 Rattus norvegicus. A single intraarticular infiltration of sodium iodoacetate (2 mg solution) through the patellar ligament induced joint damage in previously anesthetized animals. We divided the animals into three groups: a control group, a collagen peptide group, and a hydrolyzed collagen group. Treatment consisted of oral administration of collagen peptide or hydrolyzed collagen for 30 days. Afterwards, we euthanized the animals and studied the joint chondral changes. We evaluated the results according to the chondrocyte clusters count and a histological evaluation, as per Pritzker et al. Results There was no statistical significance in injury stages between the control, hydrolyzed collagen, and collagen peptide groups (p= 0.11). Regarding scores, there was a statistical significance between the groups treated with hydrolyzed collagen and collagen peptide (p< 0.05), but not in comparison with the control group. Conclusion The proposed treatments of the induced chondral lesion with the oral administration of hydrolyzed collagen or collagen peptides were effective, resulting in lesion stabilization or regression, and warranting further experimental research to understand and improve the primary outcome of this study.


Resumo Objetivo Avaliar os efeitos do colágeno hidrolisado e do peptídeo de colágeno no tratamento de lesões condrais superficiais de ratos. Método Foram utilizados 18 Rattus norvegicus nesta pesquisa. O dano articular foi induzido por uma única infiltração intra-articular de iodoacetato de sódio (solução 2 mg), injetada através do ligamento patelar da articulação dos animais previamente anestesiados. Os animais foram distribuídos em três grupos: grupo controle, grupo peptídeo de colágeno e grupo colágeno hidrolisado. O tratamento foi realizado por 30 dias com a administração via oral do peptídeo de colágeno ou do colágeno hidrolisado. Posteriormente, foi realizada a eutanásia dos experimentos e seguiu-se para o estudo das alterações condrais articulares. Os resultados foram avaliados conforme contagem de condrócitos por cluster e através da avaliação histológica segundo Pritzker et al. Resultados Ao observar os estágios de lesão, não foi observada significância estatística entre os grupos controle, colágeno hidrolisado e peptídeo de colágeno (p= 0,11). Ao observar os escores, houve significância estatística na comparação do grupo tratado com colágeno hidrolisado e o grupo peptídeo colágeno (p< 0,05), porém sem diferença estatística em relação ao grupo controle. Conclusão Os tratamentos propostos da lesão condral induzida com uso de colágeno hidrolisado ou peptídeos de colágeno via oral mostraram-se eficazes, com estabilização ou regressão da lesão apresentada em ratos, merecendo novas pesquisas experimentais com o intuito de compreender e melhorar o desfecho primário deste trabalho.


Subject(s)
Animals , Rats , Collagen , Knee Injuries/therapy
11.
Beijing Da Xue Xue Bao ; (6): 1097-1104, 2023.
Article in Chinese | WPRIM | ID: wpr-1010174

ABSTRACT

OBJECTIVE@#To investigate whether the placement of absorbable collagen membrane increase the stability of alveolar ridge contour after guided bone regeneration (GBR) using buccal punch flap.@*METHODS@#From June 2019 to June 2023, patients who underwent GBR using buccal punch flap simultaneously with a single implant placement in posterior region (from first premolar to second molar) were divided into coverage group, in which particular bone graft was covered by collagen membrane and non-coverage group. Cone beam CT (CBCT) was taken before surgery (T0), immediately after surgery (T1), and 3-7 months after surgery (T2), and the thickness of the buccal bone plate at different levels (0, 2, 4, and 6 mm) below the smooth-rough interface of the implant (BBT-0, -2, -4, -6) was mea-sured after superimposition of CBCT models using Mimics software.@*RESULTS@#A total of 29 patients, including 15 patients in coverage group and 14 patients in non-coverage group, were investigated in this study. At T0, T1, and T2, there was no significant difference in BBT between the two groups (P>0.05). At T1, BBT-0 was (2.50±0.90) mm in the coverage group and (2.97±1.28) mm in the non-coverage group, with corresponding BBT-2 of (3.65±1.08) mm and (3.58±1.26) mm, respectively. At T2, BBT-0 was (1.22±0.55) mm in the coverage group and (1.70±0.97) mm in the non-coverage group, with corresponding BBT-2 of (2.32±0.94) mm and (2.57±1.26) mm, respectively. From T1 to T2, there were no statistically significant differences in the absolute values [(0.47±0.54)-(1.33±0.75) mm] and percentages [(10.04%±24.81%)-(48.43%±18.32%)] of BBT change between the two groups. The thickness of new bone formation in the buccal bone plate from T0 to T2 ranged from (1.27±1.09) mm to (2.75±2.15) mm with no statistical difference between the two groups at all levels.@*CONCLUSION@#In the short term, the GBR using buccal punch flap with or without collagen membrane coverage can effectively repair the buccal implant bone defect. But collagen membrane coverage showed no additional benefit on alveolar ridge contour stability compared with non-membrane coverage.


Subject(s)
Humans , Cohort Studies , Retrospective Studies , Alveolar Ridge Augmentation , Collagen , Cone-Beam Computed Tomography , Bone Regeneration , Dental Implantation, Endosseous
12.
Zhonghua fu chan ke za zhi ; Zhonghua fu chan ke za zhi;(12): 911-921, 2023.
Article in Chinese | WPRIM | ID: wpr-1012298

ABSTRACT

Objective: To perform intrauterine adhesion modeling, and to investigate the repair effect of hypoxic treated bone marrow mesenchymal stem cells (BMSC) and their derived exosomes (BMSC-exo) on endometrial injury. Methods: BMSC and their exosomes BMSC-exo extracted from rats' femur were cultured under conventional oxygen condition (21%O2) or hypoxia condition (1%O2). Intrauterine adhesion modeling was performed on 40 healthy female SD rats by intrauterine injection of bacterial lipopolysaccharide after curettage. On the 28th day of modeling, 40 rat models were randomly divided into five groups, and interventions were performed: (1) NC group: 0.2 ml phosphate buffered solution was injected into each uterine cavity; (2) BMSC group: 0.2 ml BMSC (1×106/ml) with conventional oxygen culture was injected intrauterine; (3) L-BMSC group: 0.2 ml of hypoxic cultured BMSC (1×106/ml) was injected intrauterine; (4) BMSC-exo group: 0.2 ml of BMSC-exo cultured with conventional oxygen at a concentration of 500 μg/ml was injected into the uterine cavity; (5) L-BMSC-exo group: 0.2 ml hypoxic cultured BMSC-exo (500 μg/ml) was injected intrauterine. On the 14th and 28th day of treatment, four rats in each group were sacrificed by cervical dislocation after anesthesia, and endometrial tissues were collected. Then HE and Masson staining were used to observe and calculate the number of glands and fibrosis area in the endometrium. The expressions of angiogenesis related cytokines [vascular endothelial growth factor A (VEGFA) and CD31], and fibrosis-related proteins [collagen-Ⅰ, collagen-Ⅲ, smooth muscle actin α (α-SMA), and transforming growth factor β1 (TGF-β1)] in endometrial tissues were detected by western blot. Results: (1) HE and Masson staining showed that the number of endometrial glands in L-BMSC group, BMSC-exo group and L-BMSC-exo group increased and the fibrosis area decreased compared with NC group on the 14th and 28th day of treatment (all P<0.05). Noteworthily, the changes of L-BMSC-exo group were more significant than those of BMSC-exo group (all P<0.05), and the changes of BMSC-exo group were greater than those of BMSC group (all P<0.05). (2) Western blot analysis showed that, compared with NC group, the expressions of collagen-Ⅲ and TGF-β1 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group decreased on the 14th and 28th day of treatment (all P<0.05). As the treatment time went on, the expressions of fibrosis-related proteins were different. Compared with BMSC group, the expressions of collagen-Ⅲ, α-SMA and TGF-β1 in the BMSC-exo group and L-BMSC group decreased on the 28th day (all P<0.05). Moreover, the expressions of collagen-Ⅲ and TGF-β1 in L-BMSC-exo group were lower than those in BMSC-exo group on the 28th day (all P<0.05). And the expressions of collagen-Ⅰ, α-SMA and TGF-β1 in L-BMSC-exo group were lower than those in L-BMSC group on the 28th day (all P<0.05). (3) The results of western blot analysis of VEGFA and CD31 showed that, the expressions of VEGFA and CD31 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group increased on the 14th and 28th day of treatment compared with NC group (all P<0.05). Treatment for 28 days, the expressions of VEGFA and CD31 in BMSC-exo group and CD31 in L-BMSC group were higher than those in BMSC group (all P<0.05). Moreover, the expressions of VEGFA and CD31 in L-BMSC-exo group were higher than those in BMSC-exo group and L-BMSC group on the 28th day (all P<0.05). Conclusions: Treatment of BMSC and their exosomes BMSC-exo with hypoxia could promote endometrial gland hyperplasia, inhibit tissue fibrosis, and further repair the damaged endometrium in rats with intrauterine adhesion. Importantly, hypoxic treatment of BMSC-exo is the most effective in intrauterine adhesion rats.


Subject(s)
Rats , Female , Humans , Animals , Rats, Sprague-Dawley , Transforming Growth Factor beta1/metabolism , Vascular Endothelial Growth Factor A , Exosomes/metabolism , Uterine Diseases/therapy , Collagen , Hypoxia/therapy , Fibrosis , Mesenchymal Stem Cells/metabolism , Oxygen
13.
Chin. j. integr. med ; Chin. j. integr. med;(12): 791-800, 2023.
Article in English | WPRIM | ID: wpr-1010270

ABSTRACT

OBJECTIVE@#To verify the effect of Buyang Huanwu Decoction (BHD) in ameliorating erectile dysfunction (ED) after radical prostatectomy (RP).@*METHODS@#The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis. Bilateral cavernous nerve crush injury (BCNI) in rats was used to mimic the neurovascular injury occurring after RP. By the envelope method, forty rats were randomly divided into 4 groups as follows: sham (cavernous nerves exposed only), model (BCNI), low-dosage BHD [LBHD, 12.8 g/(kg·d)], and high-dosage BHD [HBHD, 51.2 g/(kg·d)] groups, 10 rats in each group, feeding for 3 weeks respectively. Erectile function was evaluated by measuring intracavernosal pressure (ICP). Changes in the histopathology of corpus cavernosum (CC) were examined by hematoxylin-eosin staining. Meanwhile, the fibrosis of CC was measured by Masson's trichrome staining and Western blot was used to detect the expressions of collagen I, transforming growth factor beta 1 (TGF- β 1) and α-smooth muscle actin (α-SMA). Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and Western blot for determining the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The oxidative stress in the CC were assessed by the superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The proteins expression of c-Jun N-terminal kinase (JNK) and c-Jun were detected by Western blot. In addition, the expression of α-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining.@*RESULTS@#The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O- β -D-glucoside, ononin, calycosin and formononetin. Compared with the model group, LBHD and HBHD treatment improved the ICP and the circumference, area, and weight of CC (P<0.05 or P<0.01). Furthermore, LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index (P<0.05 or P<0.01). LBHD and HBHD also elevated SOD and expression level of α -SMA and Bcl-2, and reduced MDA and ROS levels, as well as expression of TGF- β 1, collagen I, Bax, p-c-JNK, p-JNK in the CC compared with the model group (P<0.05 or P<0.01). The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced, whereas the α -SMA expression increased (P<0.05 or P<0.01).@*CONCLUSIONS@#BHD can improve ED of rats with BCNI, which is related to inhibiting fibrosis, apoptosis, and oxidative stress of CC. The ROS/JNK/c-Jun signaling pathway may play an important role in the process.


Subject(s)
Male , Humans , Rats , Animals , Reactive Oxygen Species , Tandem Mass Spectrometry , bcl-2-Associated X Protein , Rats, Sprague-Dawley , Erectile Dysfunction/drug therapy , Collagen , Fibrosis , Disease Models, Animal
14.
Chin. j. integr. med ; Chin. j. integr. med;(12): 980-988, 2023.
Article in English | WPRIM | ID: wpr-1010313

ABSTRACT

OBJECTIVE@#To investigate the effect of Heliotropium indicum L. (H. indicum L.) on uterine involution and its underlying mechanisms in both in vivo and in vitro study.@*METHODS@#For in vivo studies, postpartum rats were randomly divided into 2 groups (n=24 for each): control group and treated group which were orally and daily administered with ethanolic extract of H. indicum L. (250 mg/kg body weight) until day 5 of postpartum. Uteri were collected for analysis of weight, cross-sectional area, collagen cross-sectional area, and collagen content on postpartum day 1, 3, and 5 (n=8 for each) from both groups. Blood samples were collected for hepatotoxicity and 17β-estradiol (E2) measurement. For in vitro studies, the extract effects on uterine contraction at half maximum effective concentration of 2.50 mg/mL were studied in organ bath system for at least 20 min.@*RESULTS@#Uterine parameters were significantly decreased after treated with extract of H. indicum L. (P<0.05). H. indicum L. extract significantly accelerated the reduction of those parameters and significantly decreased E2 (P<0.05). The extract facilitated uterine involution with no hepatotoxicity. H. indicum L. extract significantly stimulated uterine contraction (P<0.05) and synergized with oxytocin, prostaglandin and its precursor, linoleic acid. By investigating the different sequencing of the extract with the additional stimulants (added before or after), the two showed antagonistic effects, but still showed potentiated force when compared with control (without the stimulants).@*CONCLUSIONS@#The underlying mechanisms by which H. indicum L. facilitated uterine involution might be due to reducing E2 which induces collagenase activity, leading to decreases in uterine weight and size and stimulating uterine contraction. Our study provides new findings for future drug development for facilitating uterine involution with H. indicum L.


Subject(s)
Pregnancy , Female , Rats , Animals , Heliotropium , Uterus , Plant Extracts/pharmacology , Oxytocin , Collagen/pharmacology
15.
Sheng Li Xue Bao ; (6): 179-187, 2023.
Article in Chinese | WPRIM | ID: wpr-980995

ABSTRACT

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 μmol/L) with or without Ang II (0.4 μmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.


Subject(s)
Rats , Mice , Animals , Rats, Sprague-Dawley , Angiotensin II/pharmacology , Fibroblasts , Mice, Inbred C57BL , Fibrosis , Collagen/pharmacology , Collagen Type I/metabolism , RNA, Messenger/metabolism , Myocardium/pathology
16.
Zhonghua zhong liu za zhi ; (12): 389-395, 2023.
Article in Chinese | WPRIM | ID: wpr-984734

ABSTRACT

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.


Subject(s)
Animals , Mice , Tumor Microenvironment , Coculture Techniques , Liver Neoplasms/pathology , Cadherins , Curcumin/pharmacology , Collagen , Cell Line, Tumor
17.
Zhonghua Yu Fang Yi Xue Za Zhi ; (12): 597-606, 2023.
Article in Chinese | WPRIM | ID: wpr-985450

ABSTRACT

Scarring, naturally induced by fibroblasts(Fb) during wound healing, is an essential process in response to repair damaged tissue. Excessive Fb proliferation which produces the excessive collagen deposition, including increased extracellular matrix synthesis or insufficient decomposition, typically contributes to hypertrophic scar(HS) formation. Although exact mechanisms of HS are not yet fully understood, it is generally believed that dysfunction of Fb and regulation of signal pathways play an important role in HS formation. Biologically, Fb function is affected by various factors such as cytokines, extracellular matrix and itself. In addition, modifications of miRNA, ceRNA, lncRNA, peptides and histones participate in HS formation by affecting the biological function of Fb. Despite the clinical importance, very few therapeutic modalities are available to prevent HS. To achieve this, a deeper characterization of Fb is required to identify mechanisms of HS. To the aspect of HS prevention and treatment, we review recent findings, concentrating on Fb function and collagen secretion. The objective of this article is to frame the current understanding, gain the deeper insights into Fb function, and provide the more comprehensive cognition and perspective for prevention and treatment of HS.


Subject(s)
Humans , Cicatrix, Hypertrophic/metabolism , Collagen/therapeutic use , Fibroblasts , Signal Transduction , Extracellular Matrix/metabolism
18.
Article in English | WPRIM | ID: wpr-1010687

ABSTRACT

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.


Subject(s)
Humans , Paxillin/metabolism , Protein-Lysine 6-Oxidase/metabolism , Carcinoma, Squamous Cell/pathology , Epithelial-Mesenchymal Transition , Integrin alpha2beta1/metabolism , Mouth Neoplasms/pathology , Collagen/metabolism , Fibroblasts , Extracellular Vesicles/metabolism , Cell Line, Tumor , Tumor Microenvironment
19.
Article in English | WPRIM | ID: wpr-1010711

ABSTRACT

The present study aimed to assess the molecular profiles of subepithelial connective tissue grafts (CTGs) obtained at different locations and depths in the human palate. Sixty-four CTGs belonging to anterior deep (AD), anterior superficial (AS), posterior deep (PD), and posterior superficial (PS) groups were subjected to RNA-Sequencing and their transcriptomes were analyzed computationally. Functional correlations characterizing the CTG groups were validated by cell biological experiments using primary human palatal fibroblasts (HPFs) extracted from the CTGs. A clearly more pronounced location-dependent than depth-dependent difference between the grafts, with a minimal number of genes (4) showing no dependence on the location, was revealed. Epithelial, endothelial, and monocytic cell migration was strongly (P < 0.001) potentiated by AD- and PS-HPFs. Moreover, significantly increased expression of genes encoding C-C and C-X-C motif chemokine ligands as well as significantly (P < 0.01) activated p38 signaling suggested immunomodulatory phenotype for AD- and PS-HPFs. Increased growth factor gene expression and significantly activated (P < 0.001) Erk and Akt signaling in HPFs originating from A-CTGs implied their involvement in cell survival, proliferation, and motility. Prominent collagen-rich expression profile contributing to high mechanical stability, increased osteogenesis-related gene expression, and strongly activated (P < 0.001) Smad1/5/8 signaling characterized HPFs originating from P-CTGs. The present data indicate that in humans, differences between palatal CTGs harvested from different locations and depths appear to be location- rather than depth-dependent. Our findings provide the basis for future personalization of the therapeutic strategy by selecting an optimal graft type depending on the clinical indications.


Subject(s)
Humans , Connective Tissue/transplantation , Palate , Collagen , Fibroblasts , Signal Transduction
20.
Article in Chinese | WPRIM | ID: wpr-1009093

ABSTRACT

OBJECTIVE@#To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits.@*METHODS@#hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues.@*RESULTS@#Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups.@*CONCLUSION@#Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.


Subject(s)
Pregnancy , Female , Humans , Rabbits , Animals , Vascular Endothelial Growth Factor A/metabolism , Fibronectins/metabolism , Collagen Type I/genetics , Tenascin/metabolism , Collagen/metabolism , Anterior Cruciate Ligament/surgery , Mesenchymal Stem Cells , Tendons/metabolism , Fibroblasts/metabolism
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