Collagen I and collagen II immunohistochemistry analyses on in vitro 3D poly(lactic-co-glycolic acid) seeded with intervertebral disc cells with and without fibrin scaffold

Objectives/Research Problem: Successful formation of 3D tissue constructs requires appropriate combination of the three established tissue engineering principles. The principles include quality cell source, biocompatible material scaffold and suitable biological signalling factors. This preliminary study aims to evaluate in vitro tissue constructs engineered from poly(lactic-co-glycolic acid) (PLGA) seeded with intervertebral disc (IVD) cells namely annulus fibrosus (AF), nucleus pulposus (NP), and a combination of AF:NP (1:1) with and without fibrin using specific cartilaginous markers i.e. collagen I and II immunohistochemistry staining. Materials and Method: Porous PLGA discs (7.0mm dia. X 3.0mm height) were fabricated using solvent casting and salt leaching method. The cells were harvested from rabbits’ IVD, cultured and seeded onto the pre-fabricated PLGA-based scaffolds. The resulting six “cells-scaffolds” construct groups were cultured for 3-weeks. The immunohistochemistry procedure and microscopic observation were performed at week 1, 2 and 3. Results and Discussion: Minimal cartilaginous tissue formation is noted in all constructs at week 1 until week 3. This can be appreciated by the presence of cartilage-isolated cells in lacunae embedded within extracellular matrix (ECM) ground substance. Cellular and ECM distribution are better in PLGA+Fibrin+AF:NP group than the other groups. Presence of brownish precipitation in most of the constructs after the immunolocalization of collagen I and II indicates positive results. It demonstrates that certain constructs have cartilaginous properties. Conclusion: The combination of PLGA and fibrin has the potential in facilitating early chondrogenesis of in vitro constructs engineered from AF, NP, and the combination of AF:NP (1:1) cells. The minimal cartilaginous tissue formation may be due to inefficient cells seeding. While it is apparent that human factor is unavoidable, there is much evidence in biomedical research and other fields that certain methodology can be refined to prevent errors before one’s experiment is compromised. In this case, the cells seeding method should be observed and improved for future IVD tissue engineering research

Histology staining on in vitro 3D poly(lactic-co-glycolic acid) seeded with annulus fibrosus, nucleus pulposus, and a combination of annulus fibrosus: nucleus pulposus (1:1) cells with and without fibrin scaffold

Objectives/Research Problem: Poly(lactic-co-glycolic acid) (PLGA), an FDA-approved, synthetic copolymer has been widely applied in clinical settings as one of the suturing materials. It has been used as cartilage tissue engineering scaffolds because it is bioabsorbable and safe for human use. PLGA can be prepared alone or in combination with other biomaterials such as fibrin to enhance the surface-adhesion properties. Fibrin helps to hold the cells inside the scaffolds and thus minimizes cells lost. This condition facilitates homogeneous cells distribution and extracellular matrix (ECM) production. This study aimed to evaluate in vitro constructs engineered from PLGA seeded with intervertebral disc (IVD) cells namely annulus fibrosus (AF) cell, nucleus pulposus (NP) cell, and a combination of AF:NP (1:1) with and without fibrin using histology staining. Materials and Method: All cell groups were cultured until passage 1 (P1) prior to seeding step onto the pre-fabricated PLGA scaffolds. Approx. 1.0x106 cells were used per scaffold. The resulted “cells-scaffolds” constructs were cultured for 3-weeks. The microscopic evaluation using H&E, Alcian Blue and Safranin O staining was performed on all constructs at weeks 1, 2 and 3. Results and Discussion: The overall results suggested minimal formation of cartilaginous tissue at week 1 until week 3 in all groups. Formation of cartilaginous tissue is indicated by the presence of cartilage-isolated cells in lacunae spaces. PLGA+Fibrin seeded with AF:NP demonstrated better cellular and ECM distribution than the other PLGA based constructs. Glycosaminoglycan (GAG) accumulation is noted in most constructs using Alcian Blue staining. However, the presence of proteoglycan-rich matrix was not detected in most constructs using Safranin O staining. This may be due to the immature nature of the in vitro tissue constructs. They have yet to produce cartilage specific proteoglycan-rich matrix. Conclusion: This preliminary study showed that PLGA+Fibrin has the potential to promote early formation of in vitro cartilaginous constructs engineered from the IVD cells. It is hoped that the findings provide a useful information for future research in IVD tissue engineering