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Intratendinous Injection of Hydrogel for Reseeding Decellularized Human Flexor Tendons
Chao Long, AB; Michael G. Galvez, MD; Anais Legrand, MD; Lydia-Marie Joubert, PhD; Zhen Wang, MD; Arhana Chattopadhyay, BA; James Chang, MD; Paige Fox, MD, PhD
Stanford University, Palo Alto, CA

Tendon tissue engineering addresses the challenge of limited donor tendons for tendon reconstruction. Decellularized tendons are a possible source for reconstruction, however reseeding of only the tendon surface is undesirable. We utilize a novel needle injection technique to evaluate intratendinous delivery of cells.

Decellularized human tendons (n=3/group) were reseeded with rat adipose-derived stem cells (ASCs) in culture, injected with fetal bovine serum (FBS), or with hydrogel. ASCs were stained with PKH26 dye and seeded at 0.5 million cells/mL. On Day 7, tendons were embedded and cryosectioned, cross-sections (n=30/group) were imaged under fluorescence microscopy and PKH26+ cells in cross-section were counted. Scanning electron microscopy (SEM) was used to confirm the location of cells. To evaluate cell viability, we delivered luciferase-labeled ASCs and performed bioluminescent imaging. To evaluate synthetic ability, immunohistochemistry (IHC) of procollagen was performed. ASCs' ability to attract tenocytes was assessed by seeding tenocytes in the upper chamber and either tenocytes (negative control) or ASCs in the lower chamber of a transwell plate. Cell-to-cell interaction was assessed by directly co-culturing ASCs and tenocytes at various ratios, measuring proliferation and Collagen I production, and quantifying synergy using the interaction index. Finally, tensile strength was tested.

Both FBS (p<0.001) and hydrogel (p<0.001) injection led to more ASCs inside the tendon compared to culturing. On SEM, the cultured group demonstrated cells on the surface but not inside, while both FBS and hydrogel injection groups demonstrated cells inside but not on the surface (Figure 1). Hydrogel injection initially demonstrated greater bioluminescence than culturing (p<0.005) and FBS injection (p<0.05). IHC of procollagen for injection groups demonstrated positive intratendinous staining correlating with the location of PKH26+ cells seen under fluorescence microscopy (Figure 2). ASC co-culture led to greater tenocyte migration (p<0.05). Interaction index (II) of hemocytometer counts, CyQuant assay, and Collagen I were >1 for all co-culture ratios, demonstrating synergistic proliferation and collagen production as compared to 100% ASC and 100% tenocyte controls (II=1) (p<0.05). There were no differences in tensile strength (p>0.05).

Hydrogel injection demonstrated the greatest intratendinous seeding efficiency and consistency, without compromising tensile strength. Intratendinous ASCs demonstrated synthetic capabilities. Tenocyte migration suggests that injected ASCs have the potential to attract tenocytes inside the tendon, where synergistic proliferation and Collagen I production can then promote intrinsic tendon healing. Figure 1

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