Punch shear test for quantitative evaluation of bone defect repair in Wistar rat tibia.

INTRODUCTION: Bone healing is a complex process influenced by biological and mechanical factors that restores the injured bone's load bearing function. Qualitative methods are usually employed to assess this repair process. This study presents a method of quantitative evaluation of bone repair in circular defects produced in the medial proximal metaphysis of the tibia of rats by means of punch shear tests (PST). OBJECTIVE: To describe and test a mechanical punch shear test designed to quantify over time the regeneration of bone defects produced in Wistar rats' tibiae. MATERIAL AND METHODS: In seven experimental groups, 3.2 mm in diameter and 1.5 mm deep defects were drilled on the medial surface of both proximal tibial metaphyses of the animals, just below the joint surfaces. The animals of an additional group were maintained with intact tibiae. After one, two, three, four, six, eight and 12 weeks of follow-up, the animals were sacrificed and the tibiae removed. After removing the soft tissue, the tibial proximal metaphyses were embedded in epoxy resin and cut with a diamond cutting blade, in order to obtain bone slices with equal thickness. The PST were carried out in a universal testing machine and the maximum shear forces were measured. RESULTS: The forces increased as a function of the follow-up time in the period of one to three weeks (p <0.001); from this follow-up time on, there were no statistical significant differences between the groups, including the intact tibiae. CONCLUSION: The test method proved to be suitable to quantitatively assess bone regeneration in monocortical defects produced in the proximal metaphysis of rat tibia within one and three weeks after defect production.

Hyaluronic acid and fibrin from L-PRP form semi-IPNs with tunable properties suitable for use in regenerative medicine.

Autologous leukocyte- and platelet-rich plasma (L-PRP) combined with hyaluronic acid (HA) has been widely used in local applications for cartilage and bone regeneration. The association between L-PRP and HA confers structural and rheological changes that differ among individual biomaterials but has not been investigated. Therefore, the standardization and characterization of L-PRP-HA are important to consider when comparing performance results to improve future clinical applications. To this end, we prepared semi-interpenetrating polymer networks (semi-IPNs) of L-PRP and HA and characterized their polymerization kinetics, morphology, swelling ratio, stability and rheological behavior, which we found to be tunable according to the HA molar mass (MM). Mesenchymal stem cells derived from human adipose tissue (h-AdMSCs) seeded in the semi-IPNs had superior viability and chondrogenesis and osteogenesis capabilities compared to the viability and capabilities of fibrin. We have demonstrated that the preparation of the semi-IPNs under controlled mixing ensured the formation of cell-friendly hydrogels rich in soluble factors and with tunable properties according to the HA MM, rendering them suitable for clinical applications in regenerative medicine.

Effect of Low-Intensity Pulsed Ultrasound Stimulation, Extracorporeal Shockwaves and Radial Pressure Waves on Akt, BMP-2, ERK-2, FAK and TGF-ß1 During Bone Healing in Rat Tibial Defects.

An experimental study was conducted to determine whether low-intensity pulsed ultrasound stimulation (LIPUS), extracorporeal shockwave treatment (ESWT) and radial pressure wave treatment (RPWT) modulate Akt, bone morphogenetic protein-2 (BMP-2), extracellular signal-regulated kinase-2 (ERK-2), focal adhesion kinase (FAK) and transforming growth factor-ß1 (TGF-ß1) during bone healing in rat tibial defects. Rat tibial defects were exposed to 500 shots of ESWT delivered at 0.12 mJ/mm2, 500 impulses of RPWT operated at 2.0 bar or to daily 20-min 30 mW/cm2 LIPUS. Following 1, 3 and 6 wk, bones were harvested to determine the expression and activity of Akt, BMP-2, ERK-2, FAK and TGF-ß1. Animals exposed to ultrasound were followed up to 3 wk. Protein expression and activity were unchanged following LIPUS treatment. ESWT increased Akt activity 2.11-fold (p = 0.043) and TGF-ß1 expression 9.11-fold (p = 0.016) at 1 wk and increased FAK activity 2.16-fold (p = 0.047) at 3 wk. RPWT increased FAK activity 2.6-fold (p = 0.028) at 3 wk and decreased Akt expression 0.52-fold (p = 0.05) at 6 wk. In conclusion, the protocols employed for ESWT and RPWT modulated distinct signaling pathways during fracture healing, while LIPUS standard protocol did not change the usual signaling pathways of the proteins investigated. Future studies are required to monitor osteogenesis so that the biologic meaning of our results can be clarified.

QUANTITATIVE EVALUATION OF EXPERIMENTAL BONE REGENERATION USING INDENTATION TESTS.

OBJECTIVES: To determine whether the macroindentation test can be applied to quantitatively assess bone regeneration. METHODS: A 3.2 mm diameter transverse monocortical defect was created on the medial aspect of both proximal metaphyses of the tibia of male Unib-WH rats. For the macroindentation tests, we used 5.00 mm diameter indenters with a 3.2 mm tip. Defect testing was performed 1 to 12 weeks following the surgical procedures to compare the hardness of the newly developed tissue over the 12-week study period. Additional histological, morphological and physical/chemical data were obtained by optical and electronic microscopy, Raman, and energy dispersive x-ray spectrometry (EDS). RESULTS: The mean indentation forces increased in a time-dependent manner from 4 to 12 weeks (p<0.001). Tests performed with the 5.0 mm diameter tip were not able to measure the indentation forces in the first week after the procedure. Moreover, in the second postoperative week indentation forces and the newly formed tissue within the spinal canal were greater than those measured in the fourth and eighth weeks. CONCLUSIONS: The macroindentation test can be used to quantitatively assess bone regeneration in experimental studies. The choice of indenter tip diameter should consider the study design. Level of Evidence II, Diagnostic Studies.

OBJETIVOS: Determinar se o teste de macroendentação pode ser aplicado para avaliar quantitativamente a regeneração óssea. MÉTODOS: Foi criado um defeito monocortical transversal com 3,2 mm de diâmetro na face medial de ambas as metáfises proximais da tíbia de ratos Wistar machos. Para os testes de macroendentação, empregou-se endentador com ponta de 3,2 mm e 5,0 mm de diâmetro. O teste dos defeitos foi realizado em 1 a 12 semanas depois dos procedimentos cirúrgicos, de modo que a dureza do tecido recém-formado foi comparada no período de 12 semanas do estudo. Os dados histológicos, morfológicos e físico-químicos adicionais foram obtidos por microscopia óptica e eletrônica, espectrometria Raman e EDS (espectrometria com dispersão de energia). RESULTADOS: As forças médias de endentação aumentaram de modo dependente do tempo de 4 a 12 semanas (p < 0,001). Os testes realizados com a ponta de 5 mm de diâmetro não foram capazes de medir as forças de endentação na primeira semana depois do procedimento. Além disso, na segunda semana, as forças de endentação e o tecido recém-formado no interior do canal medular foram superiores aos da quarta e oitava semanas. CONCLUSÕES: O teste de macroendentação pode ser utilizado em estudos experimentais para avaliar quantitativamente a regeneração óssea. A escolha do diâmetro da ponta do endentador deve considerar o desenho do estudo. Nível de Evidência II, Estudos Diagnósticos.

QUANTITATIVE EVALUATION OF EXPERIMENTAL BONE REGENERATION USING INDENTATION TESTS / AVALIAÇÃO QUANTITATIVA DE REGENERAÇÃO ÓSSEA EXPERIMENTAL USANDO TESTES DE INDENTAÇÃO

ABSTRACT Objectives: To determine whether the macroindentation test can be applied to quantitatively assess bone regeneration. Methods: A 3.2 mm diameter transverse monocortical defect was created on the medial aspect of both proximal metaphyses of the tibia of male Unib-WH rats. For the macroindentation tests, we used 5.00 mm diameter indenters with a 3.2 mm tip. Defect testing was performed 1 to 12 weeks following the surgical procedures to compare the hardness of the newly developed tissue over the 12-week study period. Additional histological, morphological and physical/chemical data were obtained by optical and electronic microscopy, Raman, and energy dispersive x-ray spectrometry (EDS). Results: The mean indentation forces increased in a time-dependent manner from 4 to 12 weeks (p<0.001). Tests performed with the 5.0 mm diameter tip were not able to measure the indentation forces in the first week after the procedure. Moreover, in the second postoperative week indentation forces and the newly formed tissue within the spinal canal were greater than those measured in the fourth and eighth weeks. Conclusions: The macroindentation test can be used to quantitatively assess bone regeneration in experimental studies. The choice of indenter tip diameter should consider the study design. Level of Evidence II, Diagnostic Studies.

RESUMO Objetivos: Determinar se o teste de macroendentação pode ser aplicado para avaliar quantitativamente a regeneração óssea. Métodos: Foi criado um defeito monocortical transversal com 3,2 mm de diâmetro na face medial de ambas as metáfises proximais da tíbia de ratos Wistar machos. Para os testes de macroendentação, empregou-se endentador com ponta de 3,2 mm e 5,0 mm de diâmetro. O teste dos defeitos foi realizado em 1 a 12 semanas depois dos procedimentos cirúrgicos, de modo que a dureza do tecido recém-formado foi comparada no período de 12 semanas do estudo. Os dados histológicos, morfológicos e físico-químicos adicionais foram obtidos por microscopia óptica e eletrônica, espectrometria Raman e EDS (espectrometria com dispersão de energia). Resultados: As forças médias de endentação aumentaram de modo dependente do tempo de 4 a 12 semanas (p < 0,001). Os testes realizados com a ponta de 5 mm de diâmetro não foram capazes de medir as forças de endentação na primeira semana depois do procedimento. Além disso, na segunda semana, as forças de endentação e o tecido recém-formado no interior do canal medular foram superiores aos da quarta e oitava semanas. Conclusões: O teste de macroendentação pode ser utilizado em estudos experimentais para avaliar quantitativamente a regeneração óssea. A escolha do diâmetro da ponta do endentador deve considerar o desenho do estudo. Nível de Evidência II, Estudos Diagnósticos.