Role of Hypoxia on Growth and Differentiation of Human Adipose Derived Stem Cells Grown on Silk Fibroin Scaffold Induced by Platelet Rich Plasma

Previous research has proven that 10% platelet-rich plasma (PRP) can enhance growth and differentiation of human adipose derived stem cells (hADSC) grown on silk fibroin scaffold into chondrocytes. A low oxygen concentration (hypoxia) condition is an important factor that potentially affects the ability of hADSC to grow and differentiate. The objective of this research was to analyze the difference in growth and differentiation capacity of hADSC grown on salt leached silk fibroin scaffold supplemented by 10% PRP under normoxic and hypoxic conditions. The growth capacity of the hADSC was determined by MTT assay and differentiation was tested using glycosaminoglycan (GAG) content analysis, while chondrocyte markers were visualized with the immunocytochemistry (ICC) method. This research observed hADSC proliferation under normoxic and hypoxic conditions for 21 days. Visualization of type 2 collagen showed that it was more abundant under hypoxia compared to normoxia. HIF-1α was only detected in the hADSC cultured in hypoxic conditions. In conclusion, culture under hypoxic conditions increases the capacity of hADSC to grow and differentiate into chondrocytes. This is the first study that has shown that hypoxia is able to enhance the proliferation and differentiation of hADSC grown on 3D salt leached silk fibroin scaffold supplemented by 10% PRP.

The corticospinal tract structure of collagen/silk fibroin scaffold implants using 3D printing promotes functional recovery after complete spinal cord transection in rats

No effective treatment has been established for nerve dysfunction caused by spinal cord injury (SCI). Orderly axonal growth at the site of spinal cord transection and creation of an appropriate biological microenvironment are important for functional recovery. To axially guiding axonal growth, designing a collagen/silk fibroin scaffold fabricated with 3D printing technology (3D-C/SF) emulated the corticospinal tract. The normal collagen/silk fibroin scaffold with freeze-drying technology (C/SF) or 3D-C/SF scaffold were implanted into rats with completely transected SCI to evaluate its effect on nerve repair during an 8-week observation period. Electrophysiological analysis and locomotor performance showed that the 3D-C/SF implants contributed to significant improvements in the neurogolical function of rats compared to C/SF group. By magnetic resonance imaging, 3D-C/SF implants promoted a striking degree of axonal regeneration and connection between the proximal and distal SCI sites. Compared with C/SF group, rats with 3D-C/SF scaffold exhibited fewer lesions and disordered structures in histological analysis and more GAP43-positive profiles at the lesion site. The above results indicated that the corticospinal tract structure of 3D printing collagen/silk fibroin scaffold improved axonal regeneration and promoted orderly connections within the neural network, which could provided a promising and innovative approach for tissue repair after SCI.

Treatment of cranial cruciate ligament injuries in dogs using a combination of tibial tuberosity advancement procedure and autologous mesenchymal stem cells/multipotent mesenchymal stromal cells – A pilot study

In the present pilot study, we evaluated different supplemental therapies using autologous multipotent mesenchymal stromal cells (MMSCs) for the treatment of cranial cruciate ligament defects in dogs. We used tibial tuberosity advancement (TTA) and augmented it by supportive therapy with MMSCs in three patient groups. In the first patient group, the dogs were injected with MMSCs directly into the treated stifle one month after surgery. In the second group, MMSCs were delivered in a silk fibroin scaffold which was placed in the osteotomy gap during surgery. In the third group, MMSCs were first mixed with bone tissue and blood from the patient and delivered into the osteotomy gap during surgery. In the control group, patients underwent the TTA procedure but did not receive MMSC treatment. In the group of patients who received cells in the silk fibroin scaffold during surgery, the osteotomy gap did not heal, presumably due to the low absorption of silk fibroin. Patients who received MMSCs mixed with bone tissue and blood during surgery into the osteotomy gap recovered clinically faster and had better healing of the osteotomy gap than dogs from the other two treated groups and from the control group, as assessed by clinical examination and quantification of radiographs. In conclusion, dogs that received stem cells directly into the osteotomy gap (Group 3) recovered faster compared to dogs from Groups 1 (MMSCs injected into the joint one month after surgery), 2 (cells implanted into the osteotomy gap in a silk fibroin scaffold), and the control group that did not receive additional MMSCs treatment.

Chondrogenic differentiation of Wharton’s Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma

In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin β1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.