I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. RESULTSIII. DISCUSSIONIV. CONCLUSIONSV. METHODSSUPPLEMENTARY MATERIALPeripheral nerve injuries often cause motor and sensory deficits, including atrophy of downstream muscles and loss of sensation or neuropathic pain of the innervated dermatome.11. J. A. Kouyoumdjian, Muscle Nerve 34, 785 (2006). https://doi.org/10.1002/mus.20624 Trauma to the extremities is the main cause of peripheral-nerve injuries and often attributable to traffic or industrial accidents and battlefield injuries.1,21. J. A. Kouyoumdjian, Muscle Nerve 34, 785 (2006). https://doi.org/10.1002/mus.206242. L. R. Robinson, Muscle Nerve 23, 863 (2000). https://doi.org/10.1002/(SICI)1097-4598(200006)23:6<863::AID-MUS4>3.0.CO;2-0 Consequently, peripheral nerve injuries are generally distributed among younger individuals, particularly those in their productive years.1,21. J. A. Kouyoumdjian, Muscle Nerve 34, 785 (2006). https://doi.org/10.1002/mus.206242. L. R. Robinson, Muscle Nerve 23, 863 (2000). https://doi.org/10.1002/(SICI)1097-4598(200006)23:6<863::AID-MUS4>3.0.CO;2-0 Functional recovery after microsurgical nerve repair is dependent on the distance between the trauma zone and target organ of the regenerated nerve.33. R. M. G. Menorca, T. S. Fussell, and J. C. Elfar, Hand Clin. 29, 317 (2013). https://doi.org/10.1016/j.hcl.2013.04.002 Despite considerable advancements in microsurgery, functional recovery after nerve repair can be unpredictable and incomplete.44. X. Navarro, M. Vivó, and A. Valero-Cabré, Prog. Neurobiol. 82, 163 (2007). https://doi.org/10.1016/j.pneurobio.2007.06.005 A clinical study regarding functional outcomes after repair of ulnar and median nerves revealed that only 51.6% of patients experienced return of muscle power to M4 or M5 grade, and only 42.6% reported satisfactory (S3+ to S4) sensory return.55. A. C. J. Ruijs, J.-B. Jaquet, S. Kalmijn, H. Giele, and S. E. R. Hovius, Plast. Reconstr. Surg. 116, 484 (2005). https://doi.org/10.1097/01.prs.0000172896.86594.07 Therefore, adjunct treatments for peripheral-nerve regeneration after coaptation are essential to improve functional recovery.Neurite regeneration after peripheral nerve repair is highly dependent on Schwann cells (SCs). Chromatolysis is programmed immediately after peripheral-nerve injury by degeneration of traumatized axons and their surrounding myelin. This process begins at the zone of trauma and progresses upstream to the closest node of Ranvier before extending downstream to the entire nerve in a process known as Wallerian degeneration.33. R. M. G. Menorca, T. S. Fussell, and J. C. Elfar, Hand Clin. 29, 317 (2013). https://doi.org/10.1016/j.hcl.2013.04.002 During this process, SCs phagocytose axonal and myelin debris to clean out endoneurial tubes33. R. M. G. Menorca, T. S. Fussell, and J. C. Elfar, Hand Clin. 29, 317 (2013). https://doi.org/10.1016/j.hcl.2013.04.002 and recruit macrophages to synergistically promote SC activity.33. R. M. G. Menorca, T. S. Fussell, and J. C. Elfar, Hand Clin. 29, 317 (2013). https://doi.org/10.1016/j.hcl.2013.04.002 Subsequently, emptied endoneurial tubes are refilled by aligned SCs to form bands of Büngner,66. K. Torigoe, H. F. Tanaka, A. Takahashi, A. Awaya, and K. Hashimoto, Exp. Neurology 137, 301 (1996). https://doi.org/10.1006/exnr.1996.0030 providing channels to guide the outgrowth of neurites to their target organs.66. K. Torigoe, H. F. Tanaka, A. Takahashi, A. Awaya, and K. Hashimoto, Exp. Neurology 137, 301 (1996). https://doi.org/10.1006/exnr.1996.0030 As SCs are key players in peripheral-nerve regeneration after injury, they have been proposed as an option for cell therapy during nerve coaptations.7,87. G. R. D. Evans, K. Brandt, S. Katz, P. Chauvin, L. Otto, M. Bogle, B. Wang, R. K. Meszlenyi, L. Lu, A. G. Mikos, and C. W. Patrick, Biomaterials 23, 841 (2002). https://doi.org/10.1016/S0142-9612(01)00190-98. A. Mosahebi, P. Fuller, M. Wiberg, and G. Terenghi, Exp. Neurol. 173, 213 (2002). https://doi.org/10.1006/exnr.2001.7846 However, the immunogenicity of allogenic SCs limits their use, and autologous SC transplantation is hindered by prolonged expansion in vitro and morbidities resulting from the sacrifice of donor nerves, which limits the source for harvest in clinical scenario. Other promising routes to utilize bioactive cues of SCs include transplantation of SC-like cells,9–119. Z. Rao, Z. Lin, P. Song, D. Quan, and Y. Bai, Front. Cell Neurosci. 16, 926222 (2022). https://doi.org/10.3389/fncel.2022.92622210. M. Zhang, M. H. Jiang, D.-W. Kim, W. Ahn, E. Chung, Y. Son, and G. Chi, BioMed Res. Int. 2017, 1252851. https://doi.org/10.1155/2017/125285111. X. Sun, Y. Zhu, H. Yin, Z. Guo, F. Xu, B. Xiao, W. Jiang, W. Guo, H. Meng, S. Lu, Y. Wang, and J. Peng, Stem Cell Res. Ther. 9, 133 (2018). https://doi.org/10.1186/s13287-018-0884-3 SC-derived biomaterials,9,129. Z. Rao, Z. Lin, P. Song, D. Quan, and Y. Bai, Front. Cell Neurosci. 16, 926222 (2022). https://doi.org/10.3389/fncel.2022.92622212. S. R. Cerqueira, Y.-S. Lee, R. C. Cornelison, M. W. Mertz, R. A. Wachs, C. E. Schmidt, and M. B. Bunge, Biomaterials 177, 176 (2018). https://doi.org/10.1016/j.biomaterials.2018.05.049 or augmentation of local SC recruitment.13–1513. M. Haertinger, T. Weiss, A. Mann, A. Tabi, V. Brandel, and C. Radtke, Cells 9, 163 (2020). https://doi.org/10.3390/cells901016314. L. Jiang, T. Mee, X. Zhou, and X. Jia, Stem Cell Rev. Rep. 18, 544 (2021). https://doi.org/10.1007/s12015-021-10236-515. S. Wang, C. Zhu, B. Zhang, J. Hu, J. Xu, C. Xue, S. Bao, X. Gu, F. Ding, Y. Yang, X. Gu, and Y. Gu, Biomaterials 280, 121251 (2022). https://doi.org/10.1016/j.biomaterials.2021.121251 SC-like cells require induction processes for stem cells of various origins to differentiate into SC-like cells and face the challenges to maintain their phenotype in vivo.11,1611. X. Sun, Y. Zhu, H. Yin, Z. Guo, F. Xu, B. Xiao, W. Jiang, W. Guo, H. Meng, S. Lu, Y. Wang, and J. Peng, Stem Cell Res. Ther. 9, 133 (2018). https://doi.org/10.1186/s13287-018-0884-316. M. Uz, M. Buyukoz, A. D. Sharma, D. S. Sakaguchi, S. A. Altinkaya, and S. K. Mallapragada, Acta Biomater. 53, 293 (2017). https://doi.org/10.1016/j.actbio.2017.02.018 SC-derived materials utilize the secreted factors or extracellular matrix of SCs, but may still be limited by the clinical availability of SCs, either allogenic or autologous. In addition, SCs exhibit different phenotypes (e.g., SCs of repair or mature phenotypes), and the secreted factors may work oppositely regarding axon regeneration and myelination,17,1817. P. Muangsanit, A. Day, S. Dimiou, A. F. Ata, C. Kayal, H. Park, S. N. Nazhat, and J. B. Phillips, J. Neural Eng. 17, 046036 (2020). https://doi.org/10.1088/1741-2552/abaa9c18. F. Mathot, N. Rbia, R. Thaler, A. T. Bishop, A. J. van Wijnen, and A. Y. Shin, J. Plast. Reconstr. Aesth. Surg. 73, 1473 (2020). https://doi.org/10.1016/j.bjps.2020.03.012 which require further study for elucidation. Therefore, the strategy of the present study is not focused on using exogenous SCs or related factors, but dedicated to recruit local endogenous SCs and promote their proliferation and migration in the trauma zone after microsurgical coaptation to improving nerve regeneration.Adipose-derived stem cells (ADSCs) have been proposed to enhance peripheral nerve regeneration19,2019. M.-N. Hsu, H.-T. Liao, K.-C. Li, H.-H. Chen, T.-C. Yen, P. Makarevich, Y. Parfyonova, and Y.-C. Hu, Biomaterials 140, 189 (2017). https://doi.org/10.1016/j.biomaterials.2017.05.00420. P. G. di Summa, L. Schiraldi, M. Cherubino, C. M. Oranges, D. F. Kalbermatten, W. Raffoul, and S. Madduri, Anat. Rec. 301, 1714 (2018). https://doi.org/10.1002/ar.23841 by promoting local cell activity via paracrine effects.21–2321. N. Tajiri, S. A. Acosta, M. Shahaduzzaman, H. Ishikawa, K. Shinozuka, M. Pabon, D. Hernandez-Ontiveros, D. W. Kim, C. Metcalf, M. Staples, T. Dailey, J. Vasconcellos, G. Franyuti, L. Gould, N. Patel, D. Cooper, Y. Kaneko, C. V. Borlongan, and P. C. Bickford, J. Neurosci. 34, 313 (2013). https://doi.org/10.1523/JNEUROSCI.2425-13.201422. H. Suga, J. P. Glotzbach, M. Sorkin, M. T. Longaker, and G. C. Gurtner, Ann. Plast. Surg. 72, 234 (2014). https://doi.org/10.1097/SAP.0b013e318264fd6a23. S. D. Zack-Williams, World J. Stem Cells 7, 51 (2015). https://doi.org/10.4252/wjsc.v7.i1.51 Paracrine mechanisms can involve secreted factors,2424. C. Tetta, E. Ghigo, L. Silengo, M. C. Deregibus, and G. Camussi, Endocrine 44, 11 (2013). https://doi.org/10.1007/s12020-012-9839-0 including extracellular vesicles (EVs).25,2625. H. T. Ong, S. L. Redmond, R. J. Marano, M. D. Atlas, M. von Unge, P. Aabel, and R. J. Dilley, Stem Cells Develop. 26, 405 (2017). https://doi.org/10.1089/scd.2016.020426. J. Morhayim, R. Rudjito, J. P. van Leeuwen, and M. van Driel, Curr. Mol. Biol. Rep. 2, 48 (2016). https://doi.org/10.1007/s40610-016-0034-6 EVs are proteolipid bilayer spheroids under 200 nm in diameter that can transfer intercellular signals via proteins, nucleic acids, and lipids, influencing recipient cell function.27,2827. W. P. Kuo and S. Jia, Extracellular Vesicles: Methods and Protocols ( Springer, New York, 2017).28. F. A. W. Coumans, A. R. Brisson, E. I. Buzás, F. Dignat-George, E. E. E. Drees, S. El-Andaloussi, C. Emanueli, A. Gasecka, A. Hendrix, A. F. Hill, R. Lacroix, Y. Lee, T. G. van Leeuwen, N. Mackman, I. Mäger, J. P. Nolan, E. van der Pol, D. M. Pegtel, S. Sahoo, P. R.-M. Siljander, G. Sturk, O. D. Wever, and R. Nieuwland, Circ. Res. 120, 1632 (2017). https://doi.org/10.1161/CIRCRESAHA.117.309417 ADSC-derived EVs can modify SC activity and promote neurite outgrowth via paracrine effects.13,2913. M. Haertinger, T. Weiss, A. Mann, A. Tabi, V. Brandel, and C. Radtke, Cells 9, 163 (2020). https://doi.org/10.3390/cells901016329. J. Chen, S. Ren, D. Duscher, Y. Kang, Y. Liu, C. Wang, M. Yuan, G. Guo, H. Xiong, P. Zhan, Y. Wang, H. G. Machens, and Z. Chen, J. Cell. Physiol. 122, 1 (2019). https://doi.org/10.1002/jcp.28873 Therefore, EVs may represent an alternative to cell therapy, enhancing local SC activity to promote neurite outgrowth. Conventionally, EVs are administered through systemic injection, which is challenged by rapid clearance of EVs from blood with compromised therapeutic effect;30,3130. T. Smyth, M. Kullberg, N. Malik, P. Smith-Jones, M. W. Graner, and T. J. Anchordoquy, J. Control Release 199, 145 (2015). https://doi.org/10.1016/j.jconrel.2014.12.01331. Y. Takahashi, M. Nishikawa, H. Shinotsuka, Y. Matsui, S. Ohara, T. Imai, and Y. Takakura, J. Biotechnol. 165, 77 (2013). https://doi.org/10.1016/j.jbiotec.2013.03.013 however, locally applied EVs also encounter rapid clearance and dilution by body fluid, resulting in a short halflife.32,3332. O. P. B. Wiklander, J. Z. Nordin, A. O'Loughlin, Y. Gustafsson, G. Corso, I. Mäger, P. Vader, Y. Lee, H. Sork, Y. Seow, N. Heldring, L. Alvarez-Erviti, C. E. Smith, K. L. Blanc, P. Macchiarini, P. Jungebluth, M. J. A. Wood, and S. E. Andaloussi, J. Extracell. Vesicles 4, 26316 (2015). https://doi.org/10.3402/jev.v4.2631633. T. Yamashita, Y. Takahashi, and Y. Takakura, Biol. Pharm. Bull. 41, 835 (2018). https://doi.org/10.1248/bpb.b18-00133 On the other hand, peripheral nerve regeneration requires a relatively long healing time. Furthermore, various studies have proved that sustained delivery of EVs significantly improves outcomes in vivo compared to the bolus injection of EVs.34,3534. S. Mardpour, M. H. Ghanian, H. Sadeghi-abandansari, S. Mardpour, A. Nazari, F. Shekari, and H. Baharvand, ACS Appl. Mater. Interfaces 11, 37421 (2019). https://doi.org/10.1021/acsami.9b1012635. E. A. Mol, Z. Lei, M. T. Roefs, M. H. Bakker, M. Goumans, P. A. Doevendans, P. Y. W. Dankers, P. Vader, and J. P. G. Sluijter, Adv. Healthcare Mater. 8, 1900847 (2019). https://doi.org/10.1002/adhm.201900847 Therefore, it is necessary to develop a carrier to offer steady and extended release of EVs to maintain bioactivity at the site of nerve injury and to further accelerate neurite outgrowth.Hydrogels have been proposed as carriers with the potential to mimic the extracellular matrix and promote nerve regeneration.3636. L. Huang, R. Li, W. Liu, J. Dai, Z. Du, X. Wang, J. Ma, and J. Zhao, Neural Regener. Res. 9, 1371 (2014). https://doi.org/10.4103/1673-5374.137590 In addition, tuning the hydrogel to be thermosensitive allows simple intraoperative application and rapid solidification at room temperature, ensuring that it wraps around the repaired nerve for a more focused delivery of EVs. In the literature, hydrogels have been proposed to carry a comparatively lower amount of EVs to produce and sustain the intended effect for a certain timespan3737. A. K. Riau, H. S. Ong, G. H. F. Yam, and J. S. Mehta, Front. Pharmacol. 10, 1368 (2019). https://doi.org/10.3389/fphar.2019.01368 because hydrogels prevent the loaded EVs from being cleared by body fluid or local cells prematurely.3838. B. Liu, B. W. Lee, K. Nakanishi, A. Villasante, R. Williamson, J. Metz, J. Kim, M. Kanai, L. Bi, K. Brown, G. D. Paolo, S. Homma, P. A. Sims, V. K. Topkara, and G. Vunjak-Novakovic, Nat. Biomed. Eng. 2, 293 (2018). https://doi.org/10.1038/s41551-018-0229-7 Additionally, a more focused and concentrated delivery of EVs is allowed by placing the EV-loaded hydrogel directly at the target organ. Moreover, considering that surface molecules of EVs are negatively charged,39,4039. C. Wang, M. Wang, T. Xu, X. Zhang, C. Lin, W. Gao, H. Xu, B. Lei, and C. Mao, Theranostics 9, 65 (2018). https://doi.org/10.7150/thno.2976640. A. Matsumoto, Y. Takahashi, M. Nishikawa, K. Sano, M. Morishita, C. Charoenviriyakul, H. Saji, and Y. Takakura, J. Pharm. Sci. 106, 168 (2017). https://doi.org/10.1016/j.xphs.2016.07.022 the application of a positively charged thermosensitive hydrogel would increase its affinity for ADSC-EVs, thereby facilitating a controlled and steady release. Positive charge reportedly induces and promotes SC migration, which may improve nerve regeneration.4141. S. J. Bunn, A. Lai, and J. Li, Ann. Biomed. Eng. 47, 1584 (2019). https://doi.org/10.1007/s10439-019-02259-4 Various materials provide an overall positive charge,42,4342. C. M. M. Motta, K. J. Endres, C. Wesdemiotis, R. K. Willits, and M. L. Becker, Biomaterials 218, 119335 (2019). https://doi.org/10.1016/j.biomaterials.2019.11933543. B. P. Meloni, F. L. Mastaglia, and N. W. Knuckey, Front. Neurol. 11, 286 (2020). https://doi.org/10.3389/fneur.2020.00108 including lysine, which has a high affinity for SCs4444. M. Hu, E. E. Sabelman, C. Tsai, J. Tan, and V. R. Hentz, Tissue Eng. 6, 585 (2000). https://doi.org/10.1089/10763270050199532 and neurons.4545. J. M. Zuidema, M. M. Pap, D. B. Jaroch, F. A. Morrison, and R. J. Gilbert, Acta Biomater. 7, 1634 (2011). https://doi.org/10.1016/j.actbio.2010.11.039 Furthermore, polysaccharides, including dextran, have been proven to be beneficial for peripheral-nerve regeneration by promoting neurite outgrowth.46–4846. S.-H. Chen, P.-Y. Chou, Z.-Y. Chen, D. C.-C. Chuang, S.-T. Hsieh, and F.-H. Lin, Carbohydr. Polym. 250, 116981 (2020). https://doi.org/10.1016/j.carbpol.2020.11698147. Q.-H. Gao, X. Fu, R. Zhang, Z. Wang, and M. Guo, Int. J. Biol. Macromol. 106, 749 (2018). https://doi.org/10.1016/j.ijbiomac.2017.08.07548. A. C. Pinho, M. V. Branquinho, R. D. Alvites, A. C. Fonseca, A. R. Caseiro, S. S. Pedrosa, A. L. Luís, I. Pires, J. Prada, L. Muratori, G. Ronchi, S. Geuna, J. D. Santos, A. C. Maurício, A. C. Serra, and J. F. J. Coelho, Biomater. Sci. 8, 798 (2019). https://doi.org/10.1039/C9BM00901AThus, the present study describes the design of a thermosensitive hydrogel based on a pluronic-alginate mix polymer, as a cell-free therapeutic modality to promote peripheral-nerve repair (Fig. 1). In particular, the positively charged lysine–dextran component of the hydrogel will facilitate adsorption of ADSC-EVs. This hydrogel is expected to be thermo-responsive, solidify rapidly at body temperature, and steadily release EVs locally to promote nerve regeneration.

IV. CONCLUSIONS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSIONIV. CONCLUSIONS <<V. METHODSSUPPLEMENTARY MATERIAL

This study demonstrated the therapeutic potential of cell-free therapy on peripheral-nerve regeneration by showing the effect of an EV-loaded thermosensitive hydrogel using in vitro and in vivo models. The PALDE hydrogel directly stimulated axon outgrowth and promoted SC proliferation and migration, suggesting a secondary effect on neurite outgrowth by influencing SC behavior. Moreover, the application of the PALDE hydrogel in a sciatic nerve repair model highlighted positive effects on CMAP, NCV, and muscle contraction force, along with a relative decrease in muscle atrophy. Downstream nerves in the PALDE group exhibited larger fascicle diameters and thicker myelin sheaths under TEM, consistent CMAP and NCV findings. Thus, the application of the PALDE thermosensitive hydrogel around repaired nerves provides a cell-free therapy with bioactive cues from ADSC-derived EVs to promote peripheral-nerve regeneration, representing a potentially practical design in clinical scenarios. However, the mechanism underlying the action of ADSC-EVs on neurons and SCs was not demonstrated herein. Furthermore, the underlying pathways and bioactive cues within EVs require further elucidation in future research.

V. METHODS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSIONIV. CONCLUSIONSV. METHODS <<SUPPLEMENTARY MATERIAL

A. Preparation of EVs derived from ADSCs

To collect ADSC-derived EVs, ADSC culture medium was replaced with exosome-depleted Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% exosome-depleted fetal bovine serum (FBS), 2 mM l-glutamine, and 100 U penicillin/100 U streptomycin when ADSCs reached 75%–85% confluence (passage 6–8). Supernatants were collected after 72 h of culture. EVs were isolated using size-exclusion chromatography with qEV columns (Izon, Cambridge, MA, USA) at 4 °C, following the manufacturer's protocol. Nanoparticle tracking analysis, western blot, electron microscopy, and Bradford assays were used for characterization and quantification of collected EVs (see the supplementary material).

B. Thermosensitive hydrogel preparation and examination

Pluronic F127 (P) was used as the base of the hydrogel, and alginate with or without lysine–dextran in various combinations was added to form pluronic–alginate (PA) and pluronic–alginate–lysine–dextran (PALD) hydrogels (Table S2). The hydrogels were dissolved in ddH2O at 4 °C, and calcium chloride was added. The mixtures were dispersed by gentle stirring in de-ionized water. The resulting hydrogels were stored at 4 °C for subsequent experiments. Based on the results regarding effective dose of EV (Fig. S3), the EVs (3.6 mg/ml) were mixed in a ratio of 1:2 with PA or PALD hydrogels with stirring at 4 °C for 1 h, resulting in PAE and PALDE hydrogels.

C. Characterization of thermosensitive hydrogels

1. Rheometry

The change in viscosity of P, PA, and PALD thermosensitive hydrogels with respect to temperature was quantified using an HR-2 rheometer (TA Instruments, New Castle, DE, USA) and a Peltier plate temperature system. A 40-mm steel cone geometry with a 2° angle was used for evaluation. The rate of temperature increment was 2 °C/min, with a 30 s break before each measurement, and the stress was maintained steadily at 0.1 Pa at each temperature, with angular frequency set at 1 rad/s. The storage and loss moduli were obtained during each measurement, and the ratio of the loss modulus to the angular frequency was calculated to determine the viscosity of the hydrogel.

2. Zeta potential

PA and PALD hydrogels were first diluted to 1% w/v of overall polymer concentration with de-ionized water. The pH was titrated to 7.4, and the temperature was set to 25 °C before measuring the zeta potential using a 90Plus/BI-MAS instrument (Brookhaven Instruments Corp., Holtsville, NY, USA).

3. Degradation

Degradation tests were performed in PBS (pH 7.4) solution. The initial wet weight (Wi) of 1 ml of hydrogel was measured, followed by immersion in 2 ml of PBS at 37 °C for 1 week. At predetermined times, the PBS was removed, and the wet weight of the hydrogel (Wt) was obtained. The percentage degradation was calculated using the following equation: Degradation %=Wi−WtWi×100.(1)

4. Water content

The water content of the equilibrated hydrogels was calculated based on Eq. (2), where Wswollen and Wdry represent the mass of a hydrogel in its swollen and dried states, respectively, Water content %=Wswollen−WdryWswollen×100.(2)

5. Release profile of EVs

At 37 °C, 1 ml each of solidified PAE and PALDE was immersed in 2 ml of PBS, and the solution was sampled at predetermined time points (8, 12, 24, 48, 72, and 96 h) to quantify the protein content. Bradford assays were conducted according to the manufacturer's instructions to observe the release of EVs from the hydrogels.

D. Animals

All animal experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals issued by the Animal Research Committee of the Chang-Gung Memorial Hospital (IACUC, No. 2021062401), which is in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, eighth edition, 2011). Female adult Sprague Dawley rats were used and given ad libitum access to water and food under a 12 h light/dark cycle.

E. In vitro cell experiments

1. Cell cultures

The sciatic nerves of female Sprague Dawley rats (6 weeks old) were harvested for the isolation of primary SCs. Cell culture was performed using a specific medium following the protocol described by Kaewkhaw et al.8383. R. Kaewkhaw, A. M. Scutt, and J. W. Haycock, Nat. Protoc. 7, 1996 (2012). https://doi.org/10.1038/nprot.2012.118 SCs were maintained in flasks coated with 1.5 μg/cm2 poly-L-lysine and filled with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. SCs between passages four and seven were used for in vitro experiments.Primary neurons were isolated from the dorsal root ganglion (DRG) of neonatal rats based on the protocol described by Burkey.8484. T. H. Burkey, C. M. Hingtgen, and M. R. Vasko, Methods Mol. Med. 99, 189 (2004). https://doi.org/10.1385/1-59259-770-x:189 The DRG neurons were cultured in Neurobasal-A medium (Gibco) supplemented with 2% B27, nerve growth factor (50 ng/ml), and streptomycin and penicillin (100 U/ml) at 37 °C with 5% CO2.

3. SC proliferation test

SCs were seeded in 96‐well plates (3000 cells/well) coated with 0.01% poly-L-lysine. The wells were then filled with 10% FBS DMEM plus forskolin (5 μM), N2 supplement (1% v/v), and bovine pituitary extract (20 μg/ml). PALD or PALDE hydrogel (200 μl) was placed in each well of a 24-well plate. The hydrogel was extracted with SC culture medium at 37 °C for 24 h to obtain the extraction medium. SC proliferation assays were performed in the control and experimental groups, including the PALD and PALDE groups, with 200 μl of extraction medium added to each well. After culturing for 24 and 72 h, SCs were fixed with 4% paraformaldehyde and then stained with antibodies against S100. Immunohistochemical evaluation was performed using a confocal microscope. SC proliferation was quantified using the WST-1 assay kit after culturing for 24 and 72 h. The optical density at 450 nm was measured using an ELISA (enzyme-linked immunosorbent assay) reader, and cell numbers were determined using a calibration curve.

4. SC migration assay

A gap-closure assay was performed to evaluate the efficacy of SC migration among groups using two-well silicone culture inserts (Ibidi GmbH, Martinsried, Germany) set with transparent dishes (Ibidi GmbH). The two-well inserts were first filled with 10% FBS DMEM and then seeded with SCs (1 × 104 cells/cm2) for 24 h to form a confluent monolayer, which was visible through the transparent dish. After the removal of the inserts, a 500 μm cell-free interval with clear demarcation was generated in the center of the dish. SCs were irrigated with PBS and then incubated in a serum-free medium at 37 °C. After replacing the serum-free medium with extracts of the control, PALD, and PALDE groups (in the same ratio as with the SC proliferation method), the migration of SCs from the medial edges of the interval across the 500 μm distance was observed for 48 h. Images were taken using light microscopy and digitalized (Leica QWin, Germany) after 0, 24, and 48 h of migration; data were quantified using ImageJ.

5. Neurite outgrowth assay

DRG neurons were seeded in 24-well plates (3000 cells/1.8 cm2) for 6 h until achieving quiescence. The culture medium was then replaced with extracts of the control, PALD, and PALDE groups (the medium/hydrogel ratio was 1 ml/200 μl), and neurons were cultured for 24 and 72 h. The samples were fixed with 4% paraformaldehyde and stained with antibodies against β3‐tubulin and NeuN for the visualization of neurites and cell bodies of neurons, respectively, under a confocal microscope (Leica TCS SP8X STED, Leica, Hessen, Germany). Each plate was subdivided into four quadrants, and two visual fields in each quadrant were selected for the evaluation of neuron and neurite definition and overlap. Images of the selected visual fields were captured and digitalized (Leica Imaris 3D/4D Image Visualization & Analysis software, Leica). Total neurite length was quantified using Metamorph (Molecular Devices, San Jose, CA, USA).

F. In vivo experiments

1. Sciatic nerve repair model

The sciatic nerve of an 8-week-old Sprague Dawley rat was exposed through a longitudinal lateral thigh incision under general anesthesia using isoflurane (with the flow rate at 3% for induction, followed by 2% for maintenance). The sciatic nerve was transected at a point 1.5 cm proximal to its trifurcation. Direct coaptation of the transected nerve was performed with 9–0 nylon under a surgical microscope. In the surgical control group, the wound was sutured directly after sciatic nerve coaptation. In the PALD and PALDE groups, 0.5 ml of the respective hydrogel was applied around the nerve at the coaptation site before wound closure. For in vivo experiments, eight rats were used in each group.

2. Functional outcome measurements

Three months after sciatic nerve coaptation, a second surgical procedure was performed on the rats for data collection. An IX-TA-220 recorder with integrated sensors and FT-302 force transducer (iWorx Systems Inc., Dover, NH, USA) were used to measure electroneuromyography and muscle contraction force of the repaired nerve and its motor units, following the protocols presented by Giusti8585. G. Giusti, T. Kremer, W. F. Willems, P. F. Friedrich, A. T. Bishop, and A. Y. Shin, Microsurgery 32, 35 (2011). https://doi.org/10.1002/micr.20941 and Nepomuceno.8686. A. C. Nepomuceno, E. L. Politani, E. G. da Silva, R. Salomone, M. V. L. Longo, A. G. Salles, J. d Faria, and R. Gemperli, Acta Cir. Bras. 31, 542 (2016). https://doi.org/10.1590/S0102-865020160080000007 The procedure began with exploration of the sciatic nerve of the healthy limb, and the Achilles tendon was anchored to the force transducer using a 4–0 nylon suture to measure the isometric contraction force produced by the gastrocnemius and soleus. The gastrocnemius was fitted with a miniature bipolar receiver electrode for the simultaneous recording of the CMAP. The healthy sciatic nerve examined to evaluate supramaximal CMAP; the results represent the data of normal nerves. The amplitude that produced a supramaximal CMAP was recorded for subsequent stimulation of the experimental side (usually between 1 and 3 mA with a duration of 0.1 ms). Measurements were repeated on the experimental side, and the repaired sciatic nerve was exposed. Stimulation of the repaired nerve was performed at the point 1 cm proximal to the repair site using a bipolar stimulator. Once the nerve was stimulated, CMAP, nerve conduction latency, and maximal isometric muscle contraction force measurements were obtained. The distance between the nerve stimulator and CMAP receiver was measured and divided by the nerve conduction latency to generate NCV. After the experiment, the wounds were closed using nylon sutures. The animals were euthanized using carbon dioxide chambers with the CO2 flow rate at 70%.

3. Muscle atrophy percentage

After euthanasia, the muscles of both healthy and experimental limbs were harvested from the tibia and fibula for weight measurements. The muscles harvested included the posterior compartment muscles (gastrocnemius, soleus, and tibialis posterior), anterior compartment muscles (tibialis anterior and extensor digitorum), and lateral compartment muscle (peroneus longus and brevis). The wet weight of the muscles from the experimental limb was divided by that of the healthy limb to generate the percentage of muscle atrophy.

4. Histological evaluation of downstream nerve regeneration

The quality of downstream nerve regeneration was evaluated by axon diameter and myelin thickness using TEM (Hitachi HT7800, Hitachi, Tokyo, Japan). The tibial and peroneal nerves downstream of the sciatic nerve were harvested and fixed with 3% glutaraldehyde and 2% paraformaldehyde buffered with 0.1 M cacodylate (pH 7.4) at 4 °C and then post-fixed in 1% osmium tetroxide at pH 7.4. Next, a graded series of ethanol was applied to dehydrate the samples before embedding them in EPON-812 (Electron Microscopy Sciences, Hatfield, PA, USA). Sections measuring 80 nm in thickness were obtained and stained with uranyl acetate and lead citrate. Evaluation was aimed at the region 1–1.5 cm distally to the trifurcated sciatic nerve. Images were acquired with an electron microscope (Hitachi HT7800), and myelin thickness and fascicle diameter were measured using ImageJ.

G. Statistical analysis

Data are presented as mean ± standard deviation. The means of the various groups were compared using one-way analysis of variance, and significant differences were defined by p < 0.05.

ACKNOWLEDGMENTS

This work was supported by the Chang-Gung Memorial Hospital (Grant Nos. CMRPG3L1331, CMRPG3L1332, and CORPG3M0061) and the Ministry of Science and Technology [Grant Nos. NMRPG3J0641 (108–2314-B-182A-036-), NMRPG3J0642 (109–2314-B-182A-153-), and NMRPG3J0643 (110–2314-B-182A-148-)]. We thank the Microscope Core Laboratory, Chang-Gung Memorial Hospital, Linkou, for technical assistance.

Conflict of Interest

The authors have no conflicts to disclose.

Ethics Approval

All animal experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals issued by the Animal Research Committee of the Chang-Gung Memorial Hospital (IACUC, No. 2021062401).

Author Contributions

Conceptualization: Shih-Heng Chen and Sung-Tsang Hsieh, Data curation: Shih-Heng Chen, Jing-Ru Wun, and Zhi-Yu Chen, Formal analysis: Shih-Hsien Chen and Jing-Ru Wun, Funding acquisition: Shih-Heng Chen, Hsu-Wei Fang, and Feng-Huei Lin, Investigation: Shih-Hsien Chen, Jing-Ru Wun, and Zhi-Yu Chen, Methodology: Shih-Hsien Chen and Sung-Tsang Hsieh, Project administration: Huang-Kai Kao, Pang-Yun Chou, and Zhi-Yu Chen, Resources: Shih-Heng Chen, Hsu-Wei Fang, and Feng-Huei Lin, Software: Pang-Yun Chou and Zhi-Yu Chen, Supervision: Hsu-Wei Fang, Feng-Huei Lin, and Sung-Tsang Hsieh, Validation: Shih-Hsien Chen and Jing-Ru Wun, Visualization: Shih-Hsien Chen and Jing-Ru Wun, Writing – original draft: Shih-Heng Chen, and Writing – review & editing: Shih-Heng Chen, Shih-Hsien Chen, and Feng-Huei Lin.

Shih-Heng Chen: Conceptualization (equal); Data curation (equal); Funding acquisition (equal); Resources (equal); Writing – original draft (equal); Writing – review & editing (equal). Huang-Kai Kao: Project administration (equal). Jing-Ru Wun: Data curation (equal); Formal analysis (equal); Investigation (equal); Validation (equal); Visualization (equal). Pang-Yun Chou: Project administration (equal); Software (equal). Zhi-Yu Chen: Data curation (equal); Investigation (equal); Project administration (equal); Software (equal). Shih-Hsien Chen: Formal analysis (equal); Investigation (equal); Methodology (equal); Validation (equal); Visualization (equal); Writing – review & editing (equal). Sung-Tsang Hsieh: Conceptualization (equal); Methodology (equal); Supervision (equal). Hsu-Wei Fang: Funding acquisition (equal); Resources (equal); Supervision (equal). Feng-Huei Lin: Funding acquisition (equal); Resources (equal); Supervision (equal); Writing – review & editing (equal).