Gradient hydroxyapatite nanoparticles with spatial distribution facilitate the healing of tendon-to-bone interface

Abstract

Tendon-to-bone interface (TBI) injuries have become increasingly common due to the growing competition in sports. Electrohydrodynamic (EHD) three-dimensional (3D) printing is a promising strategy for controllably fabricating biomimetic micro/ nanoscale architecture in musculoskeletal tissue engineering. This study aims to fabricate a novel biomimetic EHD-printed poly(ε-caprolactone) (PCL) with gradient hydroxyapatite (HA) nanoparticles utilizing modified dopamine self-polymerization reaction and assess the biocompatibility and efficacy of osteogenic differentiation and interface regeneration in vivo. The fabricated scaffold PCL-PDA-HA (PPH) has a diameter of 190.35 ± 41.96 nm in areas with lower HA concentration and 446.54 ± 125.42 nm in areas with higher HA concentration; the scaffold was demonstrated to profoundly facilitate osteogenic differentiation of tendon stem/progenitor cells (TSPCs), enhancing the expression of RUNX2 and ALP. On day 14, the expression of osteogenic genes, including Bmp2 (~3.12-fold, p < 0.001) and Runx2 (~3.24- fold, p < 0.001), was significantly elevated compared to those of PCL groups. New fibrocartilage formation and TBI healing were observed in the PPH group in vivo. Therefore, our work demonstrated a facile green synthesis avenue for enhancing TBI healing via TSPCs’ osteogenic differentiation, which supplied a novel approach to augment the therapeutic effects of ligament graft in TBI reconstruction.

Keywords

Gradient / Hydroxyapatite / Tendon-to-bone interface / Tendon stem/progenitor cells

Funding

This work was supported by the National Natural Science Foundation of China (No. 31971272); the International Science and Technology Cooperation Key Program project of Shaanxi Province (No.2023-GHZD-25); and the Rapid Response Project of the Air Force Military Medical University (2023KXKT081).

Conflict of interest

Jiankang He serves as the Editorial Board Member of the journal but was not involved in the editorial and peer-review process conducted for this paper, directly or indirectly. Other authors declare they have no competing interests.

References

[1] Shengnan Q, Bennett S, Wen W, et al. The role of tendon derived stem/progenitor cells and extracellular matrix components in the bone tendon junction repair. Bone. 2021; 153: 116172. doi: 10.1016/j.bone.2021.116172

[2] Derwin KA, Galatz LM, Ratcliffe A, et al. Enthesis repair: challenges and opportunities for effective tendon-to-bone healing. J Bone Joint Surg Am. 2018; 100: e109. doi: 10.2106/JBJS.18.00200

[3] Bonnevie ED, Mauck RL. Physiology and engineering of the graded interfaces of musculoskeletal junctions. Annu Rev Biomed Eng. 2018; 20: 403-429. doi: 10.1146/annurev-bioeng-062117-121113

[4] Rossetti L, Kuntz LA, Kunold E, et al. The microstructure and micromechanics of the tendon-bone insertion. Nat Mater. 2017; 16: 664-670. doi: 10.1038/nmat4863

[5] Genin GM, Thomopoulos S. The tendon-to-bone attachment: unification through disarray. Nat Mater. 2017; 16: 607-608. doi: 10.1038/nmat4906

[6] Lu HH, Thomopoulos S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu Rev Biomed Eng. 2013; 15: 201-226. doi: 10.1146/annurev-bioeng-071910-124656

[7] Yin Z, Chen X, Zhu T, et al. The effect of decellularized matrices on human tendon stem/progenitor cell differentiation and tendon repair. Acta Biomater. 2013; 9: 9317-9329. doi: 10.1016/j.actbio.2013.07.022

[8] Galatz LM, Ball CM, Teefey SA, et al. The outcome and repair integrity of completely arthroscopically repaired large and massive rotator cuff tears. J Bone Joint Surg Am. 2024; 86: 219-224. doi: 10.2106/00004623-200402000-00002

[9] Lu H, Chen C, Qu J, et al. Initiation timing of low-intensity pulsed ultrasound stimulation for tendon-bone healing in a rabbit model. Am J Sports Med. 2016; 44: 2706-2715. doi: 10.1177/0363546516651863

[10] Shi Q, Zhang T, Chen Y, et al. Local administration of metformin improves bone microarchitecture and biomechanical properties during ruptured canine achilles tendon-calcaneus interface healing. Am J Sports Med. 2022; 50: 2145-2154. doi: 10.1177/03635465221098144

[11] Jiang Q, Wang L, Liu Z, et al. Canine ACL reconstruction with an injectable hydroxyapatite/collagen paste for accelerated healing of tendon-bone interface. Bioact Mater. 2023; 20: 1-15. doi: 10.1016/j.bioactmat.2022.05.003

[12] Matsumoto T, Sato Y, Kobayashi T, et al. Adipose-derived stem cell sheets improve early biomechanical graft strength in rabbits after anterior cruciate ligament reconstruction. Am J Sports Med. 2021; 49: 3508-3518. doi: 10.1177/03635465211041582

[13] Lei T, Zhang T, Ju W, et al. Biomimetic strategies for tendon/ ligament-to-bone interface regeneration. Bioact Mater. 2021; 6: 2491-2510. doi: 10.1016/j.bioactmat.2021.01.022

[14] He SK, Ning LJ, Yao X, et al. Hierarchically demineralized cortical bone combined with stem cell-derived extracellular matrix for regeneration of the tendon-bone interface. Am J Sports Med. 2021; 49: 1323-1332. doi: 10.1177/0363546521994511

[15] Liu W, Lipner J, Xie J, et al. Nanofiber scaffolds with gradients in mineral content for spatial control of osteogenesis. ACS Appl Mater Interfaces. 2014; 6: 2842-2849. doi: 10.1021/am405418g

[16] Zhang X, Han K, Fang Z, et al. Enhancement of tendon-to-bone healing: choose a monophasic or hierarchical scaffold? Am J Sports Med. 2023; 51: 2688-2700. doi: 10.1177/03635465231182976

[17] Lee H, Dellatore SM, Miller WM, et al. Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007; 318: 426-430. doi: 10.1126/science.1147241

[18] Lee H, Rho J, Messersmith PB. Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings. Adv Mater. 2009; 21: 431-434. doi: 10.1002/adma.200801222

[19] Perikamana S, Shin YM, Lee JK, et al. Graded functionalization of biomaterial surfaces using mussel-inspired adhesive coating of polydopamine. Colloids Surf B Biointerfaces. 2017; 159: 546-556. doi: 10.1016/j.colsurfb.2017.08.022

[20] Madhurakkat PS, Lee J, Ahmad T, et al. Harnessing biochemical and structural cues for tenogenic differentiation of adipose derived stem cells (ADSCs) and development of an in vitro tissue interface mimicking tendon-bone insertion graft. Biomaterials. 2018; 165: 79-93. doi: 10.1016/j.biomaterials.2018.02.046

[21] Yang HC, Wu QY, Wan LS, et al. Polydopamine gradients by oxygen diffusion controlled autoxidation. Chem Commun (Camb). 2013; 49: 10522-10524. doi: 10.1039/c3cc46127k

[22] Ghorbani F, Zamanian A, Behnamghader A, et al. A facile method to synthesize mussel-inspired polydopamine nanospheres as an active template for in situ formation of biomimetic hydroxyapatite. Mater Sci Eng C Mater Biol Appl. 2019; 94: 729-739. doi: 10.1016/j.msec.2018.10.010

[23] Liu F, Wang X, Chen T, et al. Hydroxyapatite/silver electrospun fibers for anti-infection and osteoinduction. J Adv Res. 2020; 21: 91-102. doi: 10.1016/j.jare.2019.10.002

[24] Cai J, Wan F, Dong Q, et al. Silk fibroin and hydroxyapatite segmented coating enhances graft ligamentization and osseointegration processes of the polyethylene terephthalate artificial ligament in vitro and in vivo. J Mater Chem B. 2018; 6: 5738-5749. doi: 10.1039/c8tb01310a

[25] El-Habashy SE, Eltaher HM, Gaballah A, et al. Hybrid bioactive hydroxyapatite/polycaprolactone nanoparticles for enhanced osteogenesis. Mater Sci Eng C Mater Biol Appl. 2021; 119: 111599. doi: 10.1016/j.msec.2020.111599

[26] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials. 2006; 27: 2907-2915. doi: 10.1016/j.biomaterials.2006.01.017

[27] Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med. 2007; 13(10): 1219-1227. doi: 10.1038/nm1630

[28] Cheng P, Han P, Zhao C, et al. High-purity magnesium interference screws promote fibrocartilaginous entheses regeneration in the anterior cruciate ligament reconstruction rabbit model via accumulation of BMP-2 and VEGF. Biomaterials. 2016; 81: 14-26. doi: 10.1016/j.biomaterials.2015.12.005

[29] Hays PL, Kawamura S, Deng XH, et al. The role of macrophages in early healing of a tendon graft in a bone tunnel. J Bone Joint Surg Am. 2008; 90: 565-579. doi: 10.2106/JBJS.F.00531

[30] Qiu J, Ahn J, Qin D, et al. Biomimetic scaffolds with a mineral gradient and funnel-shaped channels for spatially controllable osteogenesis. Adv Healthc Mater. 2022; 11: e2100828. doi: 10.1002/adhm.202100828

[31] Stanton AE, Tong X, Jing SL, et al. Aligned gelatin microribbon scaffolds with hydroxyapatite gradient for engineering the bone-tendon interface. Tissue Eng Part A. 2022; 28: 712-723. doi: 10.1089/ten.TEA.2021.0099

[32] Su W, Guo J, Xu J, et al. Gradient composite film with calcium phosphate silicate for improved tendon -to-bone intergration. Chem Eng J. 2021; 404: 126473. doi: 10.1016/j.cej.2020.126473

[33] Bai L, Kasimu A, Wang S, et al. Electrohydrodynamic-printed dual-triphase microfibrous scaffolds reshaping the lipidomic profile for enthesis healing in a rat rotator cuff repair model. Small. 2024;2024:e2406069. doi: 10.1002/smll.202406069

[34] Liu X, Zhang Z, Shi Y, et al. Effect of electrohydrodynamic printing scaffold with different spacing on chondrocyte dedifferentiation. Ann Transl Med. 2022; 10: 743. doi: 10.21037/atm-22-2796

[35] Zhang X, Song W, Han K, et al. Three-dimensional bioprinting of a structure-, composition-, and mechanics-graded biomimetic scaffold coated with specific decellularized extracellular matrix to improve the tendon-to-bone healing. ACS Appl Mater Interfaces. 2023; 15: 28964-28980. doi: 10.1021/acsami.3c03793

[36] Bai L, Han Q, Meng Z, et al. Bioprinted living tissue constructs with layer-specific, growth factor-loaded microspheres for improved enthesis healing of a rotator cuff. Acta Biomater. 2022; 154: 275-289. doi: 10.1016/j.actbio.2022.10.058

[37] Chae S, Sun Y, Choi YJ, et al. 3D cell-printing of tendon-bone interface using tissue-derived extracellular matrix bioinks for chronic rotator cuff repair. Biofabrication. 2021; 13(3): 035005. doi: 10.1088/1758-5090/abd159

[38] Zheng C, Lu H, Tang Y, et al. Autologous freeze-dried, platelet-rich plasma carrying icariin enhances bone-tendon healing in a rabbit model. Am J Sports Med. 2019; 47: 1964-1974. doi: 10.1177/0363546519849657

[39] Wang J, Xu J, Wang X, et al. Magnesium-pretreated periosteum for promoting bone-tendon healing after anterior cruciate ligament reconstruction. Biomaterials. 2021; 268: 120576. doi: 10.1016/j.biomaterials.2020.120576

[40] Chien CY, Tsai WB. Poly(dopamine)-assisted immobilization of Arg-Gly-Asp peptides, hydroxyapatite, and bone morphogenic protein-2 on titanium to improve the osteogenesis of bone marrow stem cells. ACS Appl Mater Interfaces. 2013; 5: 6975-6983. doi: 10.1021/am401071f

[41] Han F, Li T, Li M, et al. Nano-calcium silicate mineralized fish scale scaffolds for enhancing tendon-bone healing. Bioact Mater. 2023; 20: 29-40. doi: 10.1016/j.bioactmat.2022.04.030

[42] Liu Q, Yu Y, Reisdorf RL, et al. Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model. Biomaterials. 2019; 192: 189-198. doi: 10.1016/j.biomaterials.2018.10.037

[43] Zuo R, Liu J, Zhang Y, et al. In situ regeneration of bone-to-tendon structures: Comparisons between costal-cartilage derived stem cells and BMSCs in the rat model. Acta Biomater. 2022; 145: 62-76. doi: 10.1016/j.actbio.2022.03.056

[44] Tarafder S, Brito JA, Minhas S, et al. In situ tissue engineering of the tendon-to-bone interface by endogenous stem/ progenitor cells. Biofabrication. 2019; 12(1): 15008. doi: 10.1088/1758-5090/ab48ca

[45] Li X, Xie J, Lipner J, et al. Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site. Nano Lett. 2009; 9: 2763-2768. doi: 10.1021/nl901582f

[46] Tanes ML, Xue J, Xia Y. A general strategy for generating gradients of bioactive proteins on electrospun nanofiber mats by masking with bovine serum albumin. J Mater Chem B. 2017; 5: 5580-5587. doi: 10.1039/C7TB00974G

[47] Xue J, Wu T, Qiu J, et al. Promoting cell migration and neurite extension along uniaxially aligned nanofibers with biomacromolecular particles in a density gradient. Adv Funct Mater. 2020; 30: 2002031. doi: 10.1002/adfm.202002031

[48] Zhu C, Pongkitwitoon S, Qiu J, et al. Design and fabrication of a hierarchically structured scaffold for tendon-to-bone repair Adv Mater. 2018;30:e1707306. doi: 10.1002/adma.201707306

[49] Yu Y, Li X, Li J, et al. , 2021, Dopamine-assisted co-deposition of hydroxyapatite-functionalised nanoparticles of polydopamine on implant surfaces to promote osteogenesis in environments with high ROS levels. Mater Sci Eng C Mater Biol Appl. 2021; 131: 112473. doi: 10.1016/j.msec.2021.112473

[50] Bolduc JA, Collins JA, Loeser RF. Reactive oxygen species, aging and articular cartilage homeostasis. Free Radic Biol Med. 2019; 132: 73-82. doi: 10.1016/j.freeradbiomed.2018.08.038

[51] Tothova L, Celec P. Oxidative stress and antioxidants in the diagnosis and therapy of periodontitis. Front Physiol. 2017 8:1055. doi: 10.3389/fphys.2017.01055

[52] Zhu C, Qiu J, Thomopoulos S, et al. Augmenting tendon-to-bone repair with functionally graded scaffolds. Adv Healthc Mater. 2021; 10: e2002269. doi: 10.1002/adhm.202002269