Modulating the phenotype and function of bone marrow-derived macrophages via mandible and femur osteoblasts.

Various studies have been investigated the phenotypic and functional distinctions of craniofacial and long bone cells involved in bone regeneration. However, the process of bone tissue regeneration after bone grafting involves complicated interactions between different cell types at the donor-recipient site. Additionally, differences in alterations of the immune microenvironment at the recipient site remained to be explored. Osteoblasts (OBs) and macrophages (MØ) play essential roles in the bone restoration and regeneration processes in the bone and immune systems, respectively. The modulation of MØ on OBs has been extensively explored in the literature, whereas limited research has been conducted on the influence of OBs on the MØ phenotype and function. In the present study, OBs from the mandible and femur (MOBs and FOBs, respectively) promoted cranial defect regeneration in rats, with better outcomes noted in the MOBs-treated group. After MOBs transplantation, a significant inflammatory response was induced, accompanied by an early increase in IL-10 secretion. And then, there was an upregulation in M2-MØ-related cell markers and inflammatory factor expression. Condition media (CM) of OBs mildly inhibited apoptosis in MØ, enhanced their migration and phagocytic functions, and concurrently increased iNOS and Arg1 expression, with MOB-CM demonstrating more pronounced effects compared to FOB-CM. In conclusion, our investigation showed that MOBs and FOBs have the ability to modulate MØ phenotype and function, with MOBs exhibiting a stronger regulatory potential. These findings provide a new direction for improving therapeutic strategies for bone regeneration in autologous bone grafts from the perspective of the immune microenvironment.

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Rabbit growth plate morphology in temporary bilateral blocking / Morfología de la placa de crecimiento de conejos durante bloqueo bilateral temporario

Blocking of the growth plate (GP) using plates with screws (tension band plating) is a modern method used to correct deformities and moderate leg length discrepancy in growing children. Determining the duration of temporary bilateral blocking without the occurrence of irreversible changes of GP is of paramount importance important. Methods: Two-month-old Californian breed male rabbits (n=30) were exposed to bilateral blocking of the distal GP of the right femur locking plates with screws for 3, 5, and 7 weeks. The fixators were removed after 5 and 7 weeks in 18 rabbits and 3 weeks after that, animals were sacri!ced. The contralateral limb was used as a control. Histological, histomorphometric, and X-ray analyses were performed. Results: During GP blocking, its height gradually decreased. This decreased was more pronounced after 7 weeks. Destructive changes progressed with an increase in the blocking duration. Three weeks after discontinuation of the bilateral blocking that lasted 5 weeks, the height of the GP signi!cantly increased 1.2 times on the lateral side and 1.9 times on the medial side (p<0.001) compared to the control. When blocking was discontinued after 7 weeks, the structure of the GP was partially restored after 3 weeks, the height of GP signi!cantly increased 1.2 times on the lateral side, and 1.07 times on the medial side (p<0.01) compared to the control. Conclusion: Restoration of the structuralfunctional features of the GP after the removal of the plates depends on the duration of temporary bilateral blocking, which must be taken into account in the clinical setting. (AU)

El bloqueo de la placa de crecimiento (PC) utilizando placas con tornillos (banda de tensión) es un método moderno utilizado para corregir deformidades y alteraciones moderadas en la longitud de las piernas en niños en crecimiento. Es de suma importancia determinar cuál debe ser la duración del bloqueo bilateral temporal sin que ocurran cambios irreversibles en la PC. Métodos: Conejos machos de raza californiana de dos meses de edad (n = 30) fueron expuestos al bloqueo bilateral de la PC distal colocando placas del fémur derecho con tornillos durante 3, 5 y 7 semanas. Los fijadores fueron retirados después de 5 y 7 semanas en 18 de los conejos, y 3 semanas después los animales fueron sacrificados. La extremidad contralateral se utilizó como control. Se realizaron análisis histológicos, histomorfométricos y de rayos X. Resultados: Durante el bloqueo de la PC, su altura disminuyó gradualmente. Esta disminución fue más pronunciada después de 7 semanas. Los cambios destructivos se incrementaron a medida aumentaba la duración del bloqueo. Tres semanas después de la interrupción del bloqueo bilateral que duró 5 semanas, la altura de la PC aumentó significativamente 1.2 veces en el lado lateral y 1.9 veces en el lado medial (p <0.001) en comparación con el control. Conclusión: La restauración de las características funcionales estructurales de la PC después de la extracción de las placas depende de la duración del bloqueo bilateral temporal, lo que debería tenerse en cuenta en el tratamiento clínico de estas alteraciones. (AU)

Osseointegration of titanium implants anodized with and without fluoride in the electrolyte: a study in rats / Osteo-integración de implantes de titanio anodizado con y sin agregado de fluoruro en el electrolito: estudio en la rata

Based on the hypothesis that fluoride acts as a bone anabolic agent, the aim of this study was to measure in rats the osseointegration of implants (grade II titanium wire, 1 mm diameter, 4 mm long) submitted to anodic oxidation in 2 M phosphoric acid solution (control implants) or b) in 2 M phosphoric acid solution plus 0.2 M NaF (F-modified implants). Chemical composition of the implants surface was assessed by energy-dispersive X-ray spectroscopy. The surface of F-modified implants contained a 2.57% fluorine in weight. Adult male Sprague Dawley rats (300-350 g body weight) received two implants (in the femur and in the tibia, close to the knee) in each hind limb. Control and F-modified implants were inserted in the left and right hind limbs, respectively. Three weeks after surgery, the animals were sacrificed. The undecalcified bones were embedded in methylmetacrylate. Sections were obtained to measure two histomorphometric magnitudes: bone-toimplant contact (BIC) and bone volume in a defined volume of tissue around the implant (BV/TV). BIC was significantly increased on F-modified implants with respect to their controls (57.2%±3.3%, vs. 47.9±3.4, p<0.05). BV/TV did not differ significantly between F-modified and control implants (24.5±2.2% vs. 22.9±1.4, p=0.30). Profiles of the average gray pixel levels of pseudo3D images showed a greater roughness of F-modified implants respect to their controls (p<0.05). The relative contributions of surface roughness and its fluorine content to the osseointegration process requires further research. (AU)

Con la hipótesis de que el ión fluoruro actúa como anabólico sobre las células óseas, el objetivo de este trabajo fue determinar el grado de osteo-integración (en la rata) de implantes (alambre de titanio II, 1 mm de diámetro, 4 mm de largo) anodizados en solución de ácido fosfórico 2 M + NaF 0,2 M (implantes-F) comparados con implantes controles, anodizados en solución de ácido fosfórico 2 M. La composición química de la superficie de los implantes fue evaluada mediante el espectro de dispersión de rayos X producidos durante la observación en el microscopio electrónico de barrido. La superficie de los implantes-F contiene 2.57% de flúor. Ratas macho Sprague-Dawley recibieron dos implantes (en el fémur y en tibia, próximos a la rodilla). Los implantes-F y controles se insertaron en las patas izquierda y derecha respectivamente. En los cortes de hueso sin decalcificación previa se midió el contacto hueso-implante (BIC) y volumen óseo en un volumen definido de tejido (BV/TV). BIC fue significativamente mayor con los Implantes-F respecto de los controles (57,2±3,3% vs. 47,9±3,4, p<0,05). BV/TV no exhibió diferencias significativas entre implantes-F y controles (24,5±2,2% vs. 22,9±1,4, p=0,30). Los perfiles de los niveles de grises de los imágenes pseudo3D de las superficies de los implantes pusieron en evidencia la mayor rugosidad de los implantes-F respecto de los controles (p<0,05). Las contribuciones relativas de la rugosidad y del flúor en el proceso de osteo-integración requieren investigación adicional. (AU)

Ischemic Stroke in Rats Enhances Bone Resorption in Vitro

We hypothesized that the formation and differentialtion of osteoclasts are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow in the early stage of stroke. We randomly divided white female Sprague-Dawley (SD) rats (n = 30) into two groups, stroke (n = 15) and sham group (n = 15). On the 7th day after stroke, after cutting away the epiphyses of the femurs and tibias, diaphyseal channels were flushed using alpha-minimum essential medium (alpha-MEM) and bone marrow cells were collected. Bone marrow stem cells, which were extracted from the femur and tibia, were cultured on the 7th day after middle cerebral artery occlusion. We then estimated the ratio of non-adherent cells to total bone marrow cells that included osteoclast precursor cells. After culturing these cells separately, cells that tested positive on the tartrate resistant acid phosphatase (TRAP) were counted and bone resorption was evaluated by using the OAAS(TM) plate. In comparison to the control group, the stroke group showed a higher increase of non-adherent cells in the hemiplegic side bone marrow. In addition, after the primary culture, the stroke group showed an increased number of TRAP positive cells and a higher degree of bone resorption estimated by OAAS(TM) plate. As a result, osteoclastogenesis and osteoclast differentiation are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow and these changes are detected as early as within the first week after middle cerebral artery occlusion in SD rats.

Comparative study of technique to obtain stem cells from bone marrow collection between the iliac crest and the femoral epiphysis in rabbits / Estudo comparativo da técnica para a obtenção de células tronco pela coleta de medula óssea entre a crista ilíaca e epífise do fêmur em coelhos

PURPOSE: To assess the technique for the collection of rabbit bone marrow stem cells from different regions to be used as an experimental model in regenerative medicine. METHODS: Thirty rabbits were allocated into 2 groups: GROUP A, n=8, animals that underwent bone marrow blood (BMB) harvesting from the iliac crest; and GROUP B: including 22 rabbits that underwent BMB harvesting from the femur epiphysis. After harvesting, mononuclear cells were isolated by density gradient centrifugation (Ficoll - Histopaque). The number of mononuclear cells per ml was counted in a Neubauer chamber and cell viability was checked through Tripan Blue method. RESULTS: Harvesting from the iliac crest yielded an average of 1 ml of BMB and 3,6.10(6) cells/ml over 1 hour of surgery, whereas an average of 3ml of BMB and 11,79.10(6) cells./ml were obtained in 30 min from the femur epiphysis with a reduced animal death rate. CONCLUSION: The analysis for the obtention of a larger number of mononuclear cells/ml from rabbit bone marrow blood was more satisfactory in the femur epiphysis than in the iliac crest.

OBJETIVO: Avaliar a técnica mais promissora para a coleta de células tronco adultas de medula óssea de coelhos para a utilização do mesmo como modelo experimental na medicina regenerativa. MÉTODOS: Foram utilizados 30 coelhos divididos em 2 grupos: GRUPO A, n=8, onde realizamos a coleta de sangue de medula óssea (MO) da crista ilíaca e grupo B, n=22, onde realizamos a coleta de sangue da medula óssea da epífise do fêmur. Após as coletas, realizamos a separação das células mononucleadas através do gradiente de densidade (Ficoll-Hystopaque). Através da câmara de Neubauer realizamos a contagem das células mononucleadas por ml. Testamos a viabilidade celular através do método Tripan Blue. RESULTADOS: Na coleta de sangue de MO na crista ilíaca obtivemos a média de 1 ml durante 1 hora de procedimento cirúrgico, obtendo a quantidade de 3,6 .10(6) células/ml, enquanto que a punção na epífise do fêmur obtivemos a média de 3 ml durante 30 minutos de procedimento cirúrgico obtendo a quantidade de 11,79.10(6) cél./ml diminuindo o óbito dos animais. CONCLUSÃO: A análise para a obtenção de maior número de células mononucleadas/ml de sangue de medula óssea de coelho foi mais satisfatória na região da epífise do fêmur em comparação com a crista ilíaca.

Biomechanics and structural adaptations of the rat femur after hindlimb suspension and treadmill running

We microscopically and mechanically evaluated the femurs of rats subjected to hindlimb unloading (tail suspension) followed by treadmill training. Female Wistar rats were randomly divided into five groups containing 12-14 rats: control I (118 days old), control II (139 days old), suspended (tail suspension for 28 days), suspended-released (released for 21 days after 28 days of suspension), and suspended-trained (trained for 21 days after 28 days of suspension). We measured bone resistance by bending-compression mechanical tests of the entire proximal half of the femur and three-point bending tests of diaphyseal cortical bone. We determined bone microstructure by tetracycline labeling of trabecular and cortical bone. We found that tail suspension weakened bone (ultimate load = 86.3 ± 13.5 N, tenacity modulus = 0.027 ± 0.011 MPa·m vs ultimate load = 101.5 ± 10.5 N, tenacity modulus = 0.019 ± 0.006 MPa·m in control I animals). The tenacity modulus for suspended and released animals was 0.023 ± 0.010 MPa·m vs 0.046 ± 0.018 MPa·m for trained animals and 0.035 ± 0.010 MPa·m for control animals. These data indicate that normal activity and training resulted in recovered bone resistance, but suspended-released rats presented femoral head flattening and earlier closure of the growth plate. Microscopically, we found that suspension inhibited new bone subperiosteal and endosteal formation. The bone disuse atrophy secondary to hypoactivity in rats can be reversed by an early regime of exercising, which is more advantageous than ordinary cage activities alone.

Estudio experimental del proceso de la reparación ósea: sus accidentes y fracasos / Experimental study of the bone repair process: its accidents and failure

Hemos llegado a comprender que en el hecho traumático, la fractura tiene dos procesos simultáneos en evolución: uno que corre por cuenta del tejido conjuntivo que producirá una cicatrización fibrosa y una reactivación de la osteogénesis que reparará con elementos propios la continuidad ósea. Esta osteogénesis no es una verdadera cicatrización ya que reproduce cuando termina en el remodelado las mismas estructuras iniciales. Cuando una fractura consolida usa para ello las células sanas que se encuentran adherentes a los extremos fracturarios haciendo osteogénesis mediante la formación de pilares que sostienen un arco de hueso plexiforme que estabiliza la fractura y separa la reparación ósea de los tejidos parostales. En el campo óptico se ve siempre a los otros elementos del tejido conjuntivo hacer una cicatrización específica del tejido conectivo que invade desde el primer momento al foco de fractura, mediante el tejido de granulación, cuyo destino es hacer fibrogénesis. En nuestros animales la consolidación osteogénica siempre se ha impuesto a partir de los extremos óseos, engloba una fibrogénesis inicial que potencialmente conduciría a la pseudoartrosis. Restos de esta fibrogénesis se observan en forma de islotes incluídos en el callo de reparación y terminan siendo sustituidos en período tardío del remodelado.

Estudio experimental del proceso de la reparación ósea: sus accidentes y fracasos / Experimental study of the bone repair process: its accidents and failure

Hemos llegado a comprender que en el hecho traumático, la fractura tiene dos procesos simultáneos en evolución: uno que corre por cuenta del tejido conjuntivo que producirá una cicatrización fibrosa y una reactivación de la osteogénesis que reparará con elementos propios la continuidad ósea. Esta osteogénesis no es una verdadera cicatrización ya que reproduce cuando termina en el remodelado las mismas estructuras iniciales. Cuando una fractura consolida usa para ello las células sanas que se encuentran adherentes a los extremos fracturarios haciendo osteogénesis mediante la formación de pilares que sostienen un arco de hueso plexiforme que estabiliza la fractura y separa la reparación ósea de los tejidos parostales. En el campo óptico se ve siempre a los otros elementos del tejido conjuntivo hacer una cicatrización específica del tejido conectivo que invade desde el primer momento al foco de fractura, mediante el tejido de granulación, cuyo destino es hacer fibrogénesis. En nuestros animales la consolidación osteogénica siempre se ha impuesto a partir de los extremos óseos, engloba una fibrogénesis inicial que potencialmente conduciría a la pseudoartrosis. Restos de esta fibrogénesis se observan en forma de islotes incluídos en el callo de reparación y terminan siendo sustituidos en período tardío del remodelado. (AU)