Functional regeneration and repair of tendons using biomimetic scaffolds loaded with recombinant periostin

Abstract

Tendon injuries disrupt the balance between stability and mobility, causing compromised functions and disabilities. The regeneration of mature, functional tendons remains a clinical challenge. Here, we perform transcriptional profiling of tendon developmental processes to show that the extracellular matrix-associated protein periostin (Postn) contributes to the maintenance of tendon stem/progenitor cell (TSPC) functions and promotes tendon regeneration. We show that recombinant periostin (rPOSTN) promotes the proliferation and stemness of TSPCs, and maintains the tenogenic potentials of TSPCs in vitro. We also find that rPOSTN protects TSPCs against functional impairment during long-term passage in vitro. For in vivo tendon formation, we construct a biomimetic parallel-aligned collagen scaffold to facilitate TSPC tenogenesis. Using a rat full-cut Achilles tendon defect model, we demonstrate that scaffolds loaded with rPOSTN promote endogenous TSPC recruitment, tendon regeneration and repair with native-like hierarchically organized collagen fibers. Moreover, newly regenerated tendons show recovery of mechanical properties and locomotion functions.

Fig. 1. Postn is highly expressed in postnatal tendon development and endogenous tendon injury repair.

a Heatmap of the 32 differentially expressed gene profiles between neonatal and 6–8-week-old tendons (n = 3 rats per group). b Immunohistochemistry of Postn expression in the P 1d and P 6w Achilles tendons (n = 3 rats per group). c, d RT-qPCR of Postn, Scx, and Mkx expression (c, n = 3 independent experiments, by two-tailed Student’s t test: ***P < 0.001, **P < 0.01) and Western blotting of Postn, Tnmd, Mkx at P 1d and P 6w (d). e Immunofluorescence staining of Postn, Sox2, and Oct4 in the rat Achilles tendon between P 1d and P 6w groups (n = 5 rats per group). f Immunofluorescence staining of Postn, Sox2, and Oct4 in normal and injured Achilles tendons at 1 week postoperatively (n = 5 rats per group). Data are presented as mean ± SD. Exact P values were given in the Source Data file.

Fig. 2. Postn promotes TSPC stemness and tenogenic differentiation potentials in early passage in vitro.

a Immunofluorescence staining (i) and semi-quantification (ii) of Ki67 in PBS- and rPOSTN-treated TSPCs (n = 5 biologically independent samples, by two-tailed Student’s t test: ***P < 0.001). b (i) CFU-F assay of PBS- and rPOSTN-treated TSPCs. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by two-tailed Student’s t test: **P < 0.01). c Western blotting of Sox2 and Oct4 protein expression in TSPCs after rPOSTN treatment at different concentrations. d (i) CFU-F assay for assessing the anti-oxidative stress capacity of PBS- and rPOSTN-treated TSPCs under exposure to H₂O₂. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by two-tailed Student’s t test: **P < 0.01). e (i) SAβ-gal staining (top panel) and immunofluorescence staining of DNA injury-related protein γ-H2AX (bottom panel) of PBS- and rPOSTN-treated TSPCs suffering from H₂O₂ stimulation. Blank: without H₂O_2. The blue cells are senescent cells. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001). f Western blotting of senescence-related protein P53, P21, and γ-H2AX of PBS- and rPOSTN-treated TSPCs suffering from H₂O₂ stimulation. g (i) Sirius Red staining (left panel) and Masson’s trichrome staining (right panel) of PBS- and rPOSTN-treated TSPCs. The pink and mazarine areas are positively stained respectively. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by one-way ANOVA with Tukey’s post hoc test: **P < 0.01, *P < 0.05). h Immunofluorescence staining (i) and semi-quantification (ii) of tenogenic markers Tnc, Col1, Tnmd, and Mkx in PBS- and rPOSTN-treated TSPCs (n = 3 biologically independent samples, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001, **P < 0.01, *P < 0.05). i (i) CFU-F assay of si NC- and si Postn-treated TSPCs. Si NC: negative control siRNA. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by two-tailed Student’s t test: **P < 0.01). j (i) Immunofluorescence staining of Ki67 of si NC- and si Postn-treated TSPCs. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by two-tailed Student’s t test: **P < 0.01). k (i) Representative images of SAβ-gal staining of si NC- and si Postn-treated TSPCs suffering from H₂O₂ stimulation. The blue cells are senescence cells. (ii) Semi-quantification of (i) (n = 4 biologically independent samples, by two-tailed Student’s t test: ***P < 0.001). l (i) Sirius Red staining (left panel) and Masson’s trichrome staining (right panel) of si NC- and si Postn-treated TSPCs (n = 4 biologically independent samples, by two-tailed Student’s t test: **P < 0.01). m (i) Immunofluorescence staining of tenogenic markers Tnc, Col1, Tnmd, and Mkx in si NC- and si Postn-treated TSPCs. (ii) Semi-quantification of (i) (n = 3 biologically independent samples, by two-tailed Student’s t test: **P < 0.01, *P < 0.05). Data are represented as mean ± SD. Exact P values were given in the Source Data file.

Fig. 3. Postn maintains TSPC phenotype and functions after long-term passage in vitro.

a Schematic of the TSPC serial passaging. b (i) CFU-F assay of PBS- and rPOSTN-treated TSPCs. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). c Immunofluorescence staining of stemness-related markers Sox2 and Oct4, and tendon stem cell marker CD146 of PBS- and rPOSTN-treated TSPCs (n = 4 biologically independent samples). d Western blotting of Sox2 and Oct4 protein levels of PBS- and rPOSTN-treated TSPCs. e (i) SAβ-gal staining (left panel) and immunofluorescence staining of DNA injury-related protein γ-H2AX (right panel) of PBS- and rPOSTN-treated TSPCs. The blue cells are senescent cells. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). f Western blotting of P21 protein levels of PBS- and rPOSTN-treated TSPCs. g (i) Sirius Red staining of PBS- and rPOSTN-treated TSPCs in the tenogenic medium after 14 days. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). h (i) Immunofluorescence staining of tenogenic markers Tnc, Col1, Tnmd, and Mkx in PBS- and rPOSTN-treated TSPCs. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). i Western blotting of Tnc, Col1, and Tnmd protein levels of PBS- and rPOSTN-treated TSPCs in tenogenic medium. Data are represented as mean ± SD. Exact P values were calculated by two-tailed Student’s t test and given in the Source Data file. ***P < 0.001, **P < 0.01.

Fig. 4. Postn promotes TSPC spheroid formation and maintains tenogenic potentials during long-term passage.

a Schematic of three-dimensional spheroid formation of TSPCs at the 10th passage. b (i) Microscopic images of three-dimensional spheroids of PBS- and rPOSTN-treated TSPCs at 3 days (left panel) and 7 days (right panel). (ii) Semi-quantification of (i) (n = 4 biologically independent samples). c Immunofluorescence staining of Sox2, Oct4, and Postn of PBS- and rPOSTN-treated TSPCs. d Schematic of the differentiation of three-dimensional spheroids. e Immunofluorescence staining of Tnmd and Ki67 of spheroids in the tenogenic medium at 3 days. f SEM microstructure of collagen fibrils of PBS- and rPOSTN-treated TSPCs. g (i) TEM nucleus of PBS- and rPOSTN-treated TSPCs. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). h, i Immunofluorescence staining of Scx, Col1, and F-actin of PBS- and rPOSTN-treated TSPCs on day 14, and Tnmd, Ki67, and Tnc on day 21. j (i) Microscopic images, (ii) HE staining, (iii) Sirius Red staining and (iv) TEM of parallel-aligned collagen structure in vitro at 21 days. Data are represented as mean ± SD. Exact P values were calculated by two-tailed Student’s t test and given in the Source Data file. ***P < 0.001.

Fig. 5. Facilitation of TSPC tenogenesis by biomimetic parallel-aligned collagen fibrils.

a Schematic of fabrication of parallel-aligned collagen fibers (ACF). b (i) SEM of 6–8-week native tendons and ACF. (ii) Corresponding TEM of (i) with longitudinal and cross sections. c (i) Immunofluorescence staining of F-actin and Tubulin of the single TSPC on randomly-aligned collagen fibers (RCF) and ACF after 6 h of culture. (ii) Semi-quantification of (i) (n = 4 biologically independent samples). d (i) SEM and (ii) HE staining of TSPCs on RCF and ACF at 14 days. e Immunofluorescence staining of Mkx and Tnmd of TSPCs on RCF and ACF on day 14. f Overview of the transplantation protocol of ACF into athymic mice. g Representative gross morphology of subcutaneous tissue formed by implantation of ACF with PBS- and rPOSTN-treated TSPCs in the 10th generation after 8 weeks. h Macroscopic view (i) and weight (ii) of newly formed tendon-like tissues (n = 5 biologically independent samples). i HE and Masson’s trichrome staining of sections of neotissues. j (i) SEM and TEM of collagen pattern of neotissues. (ii) Distribution of collagen fibril diameters of (i) (n = 5 biologically independent samples). k (i) Immunofluorescence staining of Ki67, Tnc, and Col1 of neotissues. (ii) Semi-quantification of (i) (n = 5 biologically independent samples). Data are represented as mean ± SD. Exact P values were calculated by two-tailed Student’s t test and given in the Source Data file. ***P < 0.001, **P < 0.01.

Fig. 6. Macro-, micro- and nano- structures of neotendons regenerated by ACF loaded with rPOSTN.

a (i-iii) Schematic of surgical procedure: (i) A full defect model; (ii) Grafting; (iii) Healing. (iv-x) Gross morphology of an Achilles tendon pre-surgery, surgery, and post-surgery. b HE and Masson’s trichrome staining of neotendons from different groups at 2, 4, and 8 weeks postoperatively (n = 5 rats per group). c (i) Immunofluorescence staining of CD146, Sox2, and Oct4 of neotendons of each group at 2 weeks postoperatively. (ii) Semi-quantification of (i) (n = 5 rats per group, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001, **P < 0.01). d (i) Immunofluorescence staining of Tnc and Tnmd of sections of each group at 8 weeks postoperatively. (ii) Semi-quantification of (i) (n = 5 rats per group, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001, **P < 0.01, *P < 0.05). e (i) SEM and TEM (transverse and longitudinal) of newly-formed tendon collagen fibrils of each group at 8 weeks postoperatively. (ii) Collagen fibril diameter distribution and circularity in the ACF and ACF-rP groups (n = 3 rats per group, by two-tailed Student’s t test: *P < 0.05). Data are represented as mean ± SD. Exact P values were given in the Source Data file.

Fig. 7. Functional regeneration and repair of injured tendons using ACF loaded with rPOSTN.

a (i) Footprints of normal and experimental rats from the Defect (without implants), ACF and ACF-rP groups at 1, 4, and 8 weeks postoperatively. (ii) Semi-quantification of AFI (n = 4 rats per group, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant). b T2-weighted MRI scans of regenerated Achilles tendons of each group at 1, 4, and 8 weeks postoperatively (n = 5 rats per group). White arrows: Achilles tendons. c (i) Images of dynamometer and (ii) Biomechanical properties of the regenerated tendons (failure force, stress at failure, and modulus) of each group at 8 weeks postoperatively (n = 5 rats per group, by two-tailed Student’s t test: ***P < 0.001, **P < 0.01, *P < 0.05). d (i) AFM morphology of native tendon and newly formed collagen fibrils of each group at 8 weeks postoperatively. (ii) Section analysis of single collagen fibrils in (i). e (i) Corresponding AFM property maps of native tendon and newly formed collagen fibrils of each group at postoperative week 8 in (d). (ii) Semi-quantification of Young’s modulus in (i) (n = 5 rats per group, by one-way ANOVA with Tukey’s post hoc test: **P < 0.01, ns: not significant). f (i) Micro-CT images of trabecular bone volume in calcaneus head of each group at 8 weeks postoperatively. (ii) Semi-quantification of BMD and BV/TV in (i) (n = 5 rats per group, by one-way ANOVA with Tukey’s post hoc test: ***P < 0.001, **P < 0.01, *P < 0.05). Data are represented as mean ± SD. Exact P values were given in the Source Data file.

Fig. 8. The potential role of Postn in regulating TSPC functions and tendon regeneration.

Schematic illustration shows that rPOSTN modulates TSPC multiple cellular processes, including proliferation, stemness, anti-senescence and tenogenic differentiation in vitro, and boosts structural and functional tendon regeneration by a combination of biomimetic parallel-aligned collagen scaffolds in vivo.