The current landscape of kidney transplantation faces significant challenges, particularly due to donor shortages. Regenerative medicine and bioengineered organs offer promising alternatives. One potential approach is the decellularization and recellularization of damaged kidneys, creating artificial, transplantable organs. To build an artificial kidney, it is critical to develop an extracellular matrix (ECM) as a structural scaffold. The ECM provides both physical support and biological signals to guide the cell behavior. After acellularization, the ECM acts as a scaffold for recellularization; then, new cells are seeded onto the ECM to proliferate, mainly to “rebuild" the kidney organ. The effects of silkworm extract in tissue regeneration of the skin, eye, nerves, and vessels have been studied and the influence on cell proliferation has been Known. In this study, we highlight the potential of silkworm pupa extract in enhancing ECM remodeling and cellular recruitment, offering a promising strategy for bioengineered kidney regeneration.

In this study, we first analyzed silkworm pupa extract to identify its amino acids, proteins, and growth factors. We then prepared slices from a discarded human kidney and decellularized them using a multi-step process with 2% SDS and 1% Triton-X-100. To facilitate recellularization, we applied a suspension of single renal cells extracted from a human fetus at varying concentrations of silkworm pupa extract. The recellularization efficiency of fetal kidney cells was evaluated using flow cytometry, PCR, H&E, Masson’s trichrome, and immunohistochemical (IHC) analyses.

Our findings indicate that KI-67 staining revealed the highest cell proliferation at a concentration of 0.25 µg/µL silkworm pupa extract. Compared to the control group, real-time PCR analysis demonstrated a significant upregulation of kidney-related genes AQP2, SLC2A3, and PECAM by the second week of recellularization. H&E staining showed increased cellular recruitment in ECM, with cocoon-treated samples displaying widespread cell nuclei distribution, whereas control samples exhibited significantly fewer nuclei. Trichrome staining further confirmed enhanced collagen network formation in cocoon-treated tissues, contrasting with the disorganized, sparse collagen fibers seen in control samples (p<0.05).

Immunostaining for Desmin, a key ECM component, showed significantly higher expression in cocoon-treated tissues. Additionally, vWF exhibited the highest expression among IHC markers, significantly surpassing the control group (p<0.01). Ki67-positive cells, indicative of active proliferation, were more prevalent in cocoon-treated samples. αSMA immunostaining revealed a notable presence of positive cells throughout the ECM in treated tissues, markedly exceeding the control samples. Conversely, markers CD31, CD56, and CD326 showed negative or undetectable signals via DAB staining.

Our results suggest that silkworm pupa extract plays a crucial role in enhancing kidney scaffold recellularization. The significant upregulation of AQP2, SLC2A3, and PECAM genes indicates improved functional integration of fetal renal cells into the extracellular matrix. The increased presence of cell nuclei in treated samples, as seen in H&E staining, suggests enhanced cellular recruitment, while trichrome staining confirms superior collagen network organization in cocoon-treated tissues. These findings indicate that silkworm pupa extract supports both structural and cellular remodeling.
The immunostaining results further reinforce these observations. The elevated expression of Desmin and vWF in cocoon-treated samples highlights enhanced ECM stability and vascular remodeling, crucial for developing a functional kidney scaffold. The higher Ki67 and αSMA expression suggest active cellular proliferation and mesenchymal engagement, promoting tissue regeneration. The absence of CD31, CD56, and CD326 expression indicates selective recellularization without unwanted differentiation.

Silkworm pupa extract acts as a bioactive enhancer in kidney tissue engineering, promoting cell adhesion, proliferation, and ECM remodeling. The combination of affordability, sustainability, and ethical advantages makes silkworm pupa extract a promising and inclusive innovation in regenerative medicine. Future studies should explore in vivo applications and functional assessments to validate its clinical potential.