Automated Assessment of Cell Infiltration and Removal in Decellularized Scaffolds - Experimental Study in Rabbits.

Objective Semiquantitative and automated measurement of nuclear material removal and cell infiltration in decellularized tendon scaffolds (DTSs). Method 16 pure New Zealand rabbits were used, and the gastrocnemius muscle tendon was collected bilaterally from half of these animals (16 tendons collected); 4 were kept as control and 12 were submitted to the decellularization protocol (DTS). Eight of the DTSs were used as an in vivo implant in the experimental rotator cuff tear (RCT) model, and the rest, as well as the controls, were used in the semiquantitative and automated evaluation of nuclear material removal. The eight additional rabbits were used to make the experimental model of RCT and subsequent evaluation of cellular infiltration after 2 or 8 weeks, within the DTS. Results The semiquantitative and automated analysis used demonstrated a removal of 79% of nuclear material ( p < 0.001 and power > 99%) and a decrease of 88% (p < 0.001 and power >99%) in the area occupied by nuclear material after the decellularization protocol. On cell infiltration in DTS, an increase of 256% (p < 0.001 and power >99%) in the number of cells within the DTS was observed in the comparison between 2 and 8 weeks postoperatively. Conclusion The proposed semiquantitative and automated measurement method was able to objectively measure the removal of nuclear material and cell infiltration in DTS.

Automated Assessment of Cell Infiltration and Removal in Decellularized Scaffolds - Experimental Study in Rabbits / Avaliação automatizada da infiltração e remoção celular em scaffolds descelularizados - Estudo experimental em coelhos

Abstract Objective Semiquantitative and automated measurement of nuclear material removal and cell infiltration in decellularized tendon scaffolds (DTSs). Method 16 pure New Zealand rabbits were used, and the gastrocnemius muscle tendon was collected bilaterally from half of these animals (16 tendons collected); 4 were kept as control and 12 were submitted to the decellularization protocol (DTS). Eight of the DTSs were used as an in vivo implant in the experimental rotator cuff tear (RCT) model, and the rest, as well as the controls, were used in the semiquantitative and automated evaluation of nuclear material removal. The eight additional rabbits were used to make the experimental model of RCT and subsequent evaluation of cellular infiltration after 2 or 8 weeks, within the DTS. Results The semiquantitative and automated analysis used demonstrated a removal of 79% of nuclear material (p< 0.001 and power > 99%) and a decrease of 88% (p < 0.001 and power >99%) in the area occupied by nuclear material after the decellularization protocol. On cell infiltration in DTS, an increase of 256% (p < 0.001 and power >99%) in the number of cells within the DTS was observed in the comparison between 2 and 8 weeks postoperatively. Conclusion The proposed semiquantitative and automated measurement method was able to objectively measure the removal of nuclear material and cell infiltration in DTS.

Resumo Objetivo Mensuração semiquantitativa e automatizada da remoção de material nuclear e da infiltração celular em scaffolds tendinosos descelularizados (STDs). Método Foram utilizados 16 coelhos Nova Zelândia puros, sendo o tendão do músculo gastrocnêmio coletado bilateralmente de metade destes animais (16 tendões coletados); 4 foram mantidos como controle e 12 foram submetidos ao protocolo de descelularização (STD). Dos STDs, 8 foram utilizados como implante in vivo no modelo experimental de lesão do manguito rotador (LMR) e os restantes, assim como os controles, foram utilizados na avaliação semiquantitativa e automatizada da remoção de material nuclear. Os oito coelhos adicionais foram utilizados na confecção do modelo experimental de LMR e posterior avaliação da infiltração celular após 2 ou 8 semanas, dentro do STD. Resultados A análise semiquantitativa e automatizada utilizada demonstrou uma remoção de 79% do material nuclear (p< 0,001 e poder > 99%) e uma diminuição de 88% (p< 0,001 e poder > 99%) na área ocupada por material nuclear após o protocolo de descelularização. Sobre a infiltração celular no STD, foi observado um aumento de 256% (p< 0,001 e poder > 99%) no número de células dentro do STD na comparação entre 2 e 8 semanas de pós-operatório. Conclusão O método de mensuração semiquantitativo e automatizado proposto foi capaz de mensurar objetivamente a remoção de material nuclear e a infiltração celular no STD.

Treatment of Muscle Injury with Stem Cells - Experimental Study in Rabbits.

Objective Histological and macroscopic evaluation of the healing process of acute lesions of the femoral rectus muscle using stem cells derived from adipose tissue-derived stem cells (ADSCs). Method An experimental study was conducted with 18 hind legs of New Zealand rabbits, which were divided into three study groups according to the intervention to be performed. In group I, no surgical procedure was performed; in group II-SHAN, the experimental lesion was performed without any additional intervention protocol; in group III-Intervention, the addition of ADSCs was performed in the same topography of the experimental lesion. After the proposed period, 2 weeks, the material was collected and submitted to macroscopic and histological evaluation. Results The quantitative analysis showed that the addition of ADSCs is related to the reduction of inflammatory cells in the 2-week evaluation (164.2 cells in group II - SHAN to 89.62 cells in group III - ADSC). The qualitative analysis of the slides with Picrosirius red, noticed an increase in orange/yellow fibers in group III - ADSC, which evidences a final healing process. The macroscopic evaluation found no difference between the groups. Conclusion The use of ADSCs in the treatment of acute muscle injury presented histological advantages when compared to their non-use.

Treatment of Muscle Injury with Stem Cells - Experimental Study in Rabbits / Tratamento da lesão muscular com células-tronco - Estudo experimental em coelhos

Abstract Objective Histological and macroscopic evaluation of the healing process of acute lesions of the femoral rectus muscle using stem cells derived from adipose tissue-derived stem cells (ADSCs). Method An experimental study was conducted with 18 hind legs of New Zealand rabbits, which were divided into three study groups according to the intervention to be performed. In group I, no surgical procedure was performed; in group II—SHAN, the experimental lesion was performed without any additional intervention protocol; in group III—Intervention, the addition of ADSCs was performed in the same topography of the experimental lesion. After the proposed period, 2 weeks, the material was collected and submitted to macroscopic and histological evaluation. Results The quantitative analysis showed that the addition of ADSCs is related to the reduction of inflammatory cells in the 2-week evaluation (164.2 cells in group II - SHAN to 89.62 cells in group III - ADSC). The qualitative analysis of the slides with Picrosirius red, noticed an increase in orange/yellow fibers in group III - ADSC, which evidences a final healing process. The macroscopic evaluation found no difference between the groups. Conclusion The use of ADSCs in the treatment of acute muscle injury presented histological advantages when compared to their non-use.

Resumo Objetivo Avaliação histológica e macroscópica do processo de cicatrização das lesões agudas do músculo reto femoral, com utilização de células-tronco derivadas de tecido adiposo (ADSCs, na sigla em inglês). Método Foi realizado um estudo experimental com 18 patas traseiras de coelhos Nova Zelândia, que foram divididos em três nos grupos de estudo de acordo com a intervenção a ser realizada. No grupo I não foi realizado procedimento cirúrgico; no grupo II - SHAN foi realizado a lesão experimental sem nenhum protocolo de intervenção adicional; e no grupo III - Intervenção foi realizado a adição de ADSCs na mesma topografia onde foi realizada a lesão experimental. Após o período proposto, 2 semanas, o material foi coletado, submetido a avaliação macroscópica e histológica. Resultados A análise quantitativa demonstrou que a adição de ADSCs está relacionada com a diminuição de células inflamatórias na avaliação com 2 semanas (164,2 células no grupo II - SHAN para 89,62 células no grupo III - ADSC). A análise qualitativa das lâminas coradas com Picrosírius red demonstrou um aumento das fibras de cor laranja/amarela no grupo III - ADSC, o que evidencia um processo final de cicatrização. A avaliação macroscópica não encontrou diferença entre os grupos. Conclusão A utilização de ADSCs no tratamento de lesão muscular aguda apresentou vantagens histológicas quando comparada a sua não utilização.

Lipidomics of mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs) and skeletal muscle-derived stem cells (MDSCs), are potential sources for cell-based therapeutic strategies. However, there is little knowledge about the lipid composition of these stem cells and the mechanisms of their differentiation. Lipids have important biological and physiological functions that are critical for understanding the regulation and control of stem cell fate. This study sought to analyze the lipidome of rabbit ADSCs and MDSCs and their adipogenic and osteogenic differentiation. The MSCs were isolated and were characterized by flow cytometry. Lipids were extracted from both MSCs and differentiated cells, and the lipids were subsequently analyzed with a hybrid triple quadrupole time-of-flight mass spectrometer. The results showed a total of 1687 lipid species. MSCs exhibited different lipid profiles as well as changes in lipid composition after differentiation. Furthermore, the expression levels of N-acyl-phosphatidylethanolamine (NAPE) 54:7+NH4 (-FA 17:0(NH4)) and phosphatidylcholine (PC) 42:6+Na were higher in the adipogenic lineages in of both MSC types, and NAPE 58:2+NH4 (-FA 17:0 (NH4)) and NAPE 56:2+NH4 (-FA 17:0 (NH4)) had higher levels in the osteogenic lineages, suggesting lipid similarities in cells differentiated from different stem cell sources.

Biomechanical evaluation of tendon regeneration with adipose-derived stem cell.

The use of Adipose-Derived Stem Cells (ADSC) has been presented as a new alternative for tendon reconstruction. Have been admitted that ADSCs are related to better outcomes when used in tendon healing. This research was designed to apply the potential of ADSCs in tendon healing. Flexor digitorum superficialis tendon lesion was performed on both legs of eleven New Zealand rabbits and them, at the same time, treated as follows: Suture alone (Group III - Suture, n:10), suture associated with ADSC (Group IV - Suture + ADSC, n:10) or without suture (Group II - SHAN, n:2). At four weeks after the tendon surgery, the animal was euthanized, and the tendon evaluated (biomechanically and macroscopically). We used 5 additional New Zealand rabbits in the control group "Group I - Control, n:10". In the macroscopic evaluation, the group with ADSC presented a more homogeneous gross morphology compared with the group III. Biomechanical testing showed a lower ultimate tensile load, stiffness and a higher cross-sectional area in the group III and IV compared with the control group. The group with ADSC showed a greater ultimate tensile load, a larger cross-sectional area and bigger deformation at the ultimate tensile load when compared to the group without ADSC. In general terms, the use of ADSCs in tendon healing have biomechanical advantages compared to the non-use of ADSCs at 4 weeks after surgery. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1281-1286, 2019.