Cutting muscle for bone regeneration (割肉修骨)

Cutting muscle for bone regeneration (割肉修骨) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Cutting muscle for bone regeneration (割肉修骨) Zhengke Wang, Xiaoyang Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7135552/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As gold standard for large-sized bone defects regeneration, autologous bone transplantation has always faced the difficulty of insufficient donors. Moreover, most of the well-designed biomaterials could only achieve 20-50% repair rate. Herein, we innovatively proposed the strategy of "Cutting muscle for bone regeneration". Considering the hierarchical structure of muscle was similar to collagen in bone, we prepared hierarchical mineralized muscle for bone regeneration. Based on the unfolding of proteins induced by guanidine hydrochloride, this strategy fully utilized the complexation of troponin with Ca2+, as well as the spatial structure of interlaced arrangement of thick and thin filaments, ultimately achieved the striped mineral distribution within myofibrils. Surprisingly, the mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Compared with bone, muscle is easy to regenerate, and bone defect regeneration can be achieved by mineralized muscle, which can solve the difficulty of insufficient autologous bone donors. Physical sciences/Materials science/Biomaterials/Bioinspired materials Physical sciences/Materials science/Biomaterials/Biomineralization Physical sciences/Engineering/Biomedical engineering hierarchical structure muscle biomineralization bone regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bone, a highly mineralized tissue with complex hierarchical structure, is responsible for bearing loads, protecting fragile organs, maintaining hematopoietic and mineral balance 1-3 . Through the regulation of osteoblasts and osteoclasts, bone maintains a dynamic balance of bone resorption and bone remodeling. Thus, small-sized bone defects can self-heal through continuous bone renewal without external intervention as long as no dislocation occurs 4 . However, clinically, large bone defect that exceed critical size due to disease or trauma is still a major challenge. According to statistics, more than 20 million people worldwide suffer from bone defects every year, and about 60% of them need bone grafting to promote bone repair 5 . Although autograft still serves as the gold standard for bone defect repair, it also faces the disadvantages of limited donors 6,7 . Therefore, numerous researches have been devoted to developing biodegradable scaffolds for the repair of large-size bone defects. Traditionally, Ca-P mineral scaffolds appear to be preferred for their similar composition with bone and excellent osteoinductivity 8-11 . Of course, many researchers found that some inorganic ions such as Cu 12,13 , Zn 12,14 , Mg 15-17 , Se 18 , Sr 14 , Ce 19 , Si 20-22 etc. could also accelerate bone regeneration. Further, not limited to the change of components, the design of anisotropic structure and surface topography is also one of the research hotspots 23-32 . To date, researchers have tried various strategies including hyperthermia 33-37 , electrotherapy 38 , magnetic therapy 39,40 , immunomodulation 41-48 , cells 20,49,50 or growth factors 51-59 loading to promote the repair of large bone defects. The concept of BV/TV (bone volume/tissue volume) also was introduced to quantitatively assess the effect of bone regeneration. Regrettably, most of these well-designed scaffolds only achieve 20-50% repair rate after implantation for 8 or 12 weeks (Supplementary Table 1). Facing the dilemma of poor bone repair effect, drawing inspiration from nature seems to bring a new way. Researches are dedicated to studying the structure of natural bones from micro to macro level 60 . On the basis of previous researches, Roland Kröger et al. further subdivided the skeletal structure into 12 hierarchies 61 . Given the important role of collagen as organic templates in bone formation, many endeavors to reveal the intrafibrillar or extrafibrillar mineralization process have been made 62-65 . In fact, these studies mainly focused on the mechanism of collagen fiber mineralization in vitro. However, building complex hierarchies of collagen in vitro, similar to that in natural bone, is still difficult to achieve. Interestingly, as shown in Fig.1, the hierarchical structure of skeletal muscle (myofibril - muscle fiber - muscle fascicle - skeletal muscle) is highly similar to that of collagen (collagen microfiber - collagen fiber - collagen fiber bundle) in bone 66,67 . It is well known that collagen fibers exhibit a characteristic 67nm streak, due to the cross-arrangement of protocollagen. Correspondingly, the staggered arrangements of thick filaments and thin filaments lead to the light and dark stripes (2 μm). Bionics from natural bone, firstly create an organic template (hierarchical collagen fibers), then mineralize to form hydroxyapatite with ordered structures. Interestingly, is it feasible to select hierarchical muscle fibers as a mineralization template instead of collagen fibers, and then achieve excellent bone regeneration? Herein, “cutting muscle for bone regeneration” is validated in this research. Surprisingly, after skeletal muscle treated with guanidine hydrochloride, mineralized hydroxyapatite is striped arranged, and perpendicular to the direction of the myofibril. More importantly, this mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Compared with bone, muscle is easy to regenerate, and bone defect regeneration can be achieved by mineralized muscle, which can solve the difficulty of insufficient autologous bone donors. Replacing collagen fibers with muscle fibers, studying the mineralization mechanism of hydroxyapatite, and preparing biomimetic materials with hierarchical ordered structure, similar to natural bone. "Cutting muscle for bone regeneration" is a good combination of biomimetic materials science and practical applications, which provides a new approach for orthopedic clinical practice. Results and Discussion Fresh pork tenderloin was cut into slices, and soaked in acetone overnight to remove lipids. After degreasing, the acetone in muscle was exchanged by deionized water. Then, muscle slices were treated with deionized water (DI), guanidine hydrochloride solution (GH, 2M) and sodium hydroxide solution (NaOH, 5wt%) for 3h, respectively, and named DI group, GH group, and NaOH group. Cyclic mineralization of muscles was then taken by immersion in calcium chloride solution, sodium dihydrogen phosphate solution, and alkaline water in sequence, and terminated as the mineral content reached around 20wt%, lyophilized. Pretreatment and mineralization processes of individual muscle fibers were recorded in Movies S1-S3 (S1 for NaOH group, S2 for GH group, and S3 for DI group). Compared with the initial state, introduction of guanidine hydrochloride made muscle fibers swollen and loose, while sodium hydroxide had a stronger effect. After immersion in Ca 2+ solution, the muscle fiber exhibited obvious expansion along the cross-section (Movie S4), attributed to the complexation of Ca 2+ and troponin. After introducing calcium ions and phosphate ions successively, the muscle fibers were immersed in alkaline water, and expanded first and then contracted. Contraction in collagen fibrils by mineralization was discovered by Peter Fratzl 68 , and this is the same reason for the contraction of muscle fibers during mineralization. Mineral in muscle existed in the form of hydroxyapatite regardless of how it was pre-treated (Fig. S1 and S2), and (002), (121) crystal planes of HAP were also observed on the selected electron diffraction pattern (Fig. S3). Indeed, hydroxyapatite in muscle was polycrystalline with low crystallinity. Ca and P were uniformly distributed in mineralized muscle fibers (Cross-section, Fig. S4). At the mesoscopic scale, there was no obvious difference in CT scans of the mineralized muscles with three different pretreatment methods (Fig. S5). However, at a microscopic scale, mineral distribution emerged obvious differences in DI, GH, and NaOH groups (Fig. 2). There were distinctive light and dark stripes in pure muscle fibers, sarcomere and edge were clear (Fig. 2Ai). Correspondingly, the mineral phases with long acicular crystals were preferentially arranged along the direction of muscle fibers in DI group (Fig. 2Aii), and distributed in the space between myofibril (Fig. S6C). While muscle pretreated with guanidine hydrochloride, myofibril became fluffier, and myotome remained clear, but the edge became frizz (Fig. 2Bi). Surprisingly, the mineral phases appeared inside the myofibril (Fig. S6A), and showed a streaky distribution perpendicular to the direction of muscle fibers, with mineralized streaks spaced 1-2 microns apart (Fig. 2Bii). Small-angle diffraction pattern also indicated ordered stripe mineralization for GH group (Fig. 3D), which was not seen in pure muscle (Fig. 3C), DI group (Fig. S7A) and NaOH group (Fig. S7B). Compared with guanidine hydrochloride, a strong alkaline environment seemed to have significantly stronger disaggregation effect on muscle protein 69,70 . Myofibril structure was damaged by alkali treatment (Fig. 2Ci): myofibrils seemed to adhere together, and myotome structure was difficult to distinguish. Therefore, no matter from which angle, the mineral phases exhibited disorder distribution in NaOH group (Fig. 2Cii and Fig. S6B). Interestingly, why minerals were orderly arranged in GH group? Muscle protein was unfolded by guanidine hydrochloride 71,72 , and C-N/C-O signals after mineralization shifted from 286.06 eV to 285.35 eV, compared with pure muscle (Fig. 3). It was worth noting that the P2p signals in pure muscle, 134.02 eV and 133.07 eV, were derived from the remaining ATP-like substance, rather than calcium phosphate minerals. While P2p 1/2 and P2 3/2 located at 132.56 and 131.61 eV, respectively, for GH group. And O1s with a signal peak at 534.95eV for GH group, was not seen in any other group, and also accompanied by a decrease in the intensity of O=C peak (Fig. 3, Fig.S8). Considering the strong complexation of troponin to Ca 2+ during muscle contraction 73,74 and the striate distribution of minerals at the bright region of sarcomere in GH group (Fig.2 Bii), it was reasonable to speculate that Ca 2+ strongly ligated with carboxyl site of troponin during mineralization, and induced mineral growth as a nucleus. Guanidine hydrochloride changed the conformation of muscle protein 72 , which made the myotome structure less dense (Fig.2 Bi), and exogenous Ca 2+ could enter the interior of myotome and complex with troponin. Differently, the dense sarcomere structure in DI group prevented the traverse of Ca 2+ (Fig.2 Ai), and calcium phosphate mineral was forced to nucleate and grow in the interstitial space of myofibril. Thus, HAP long crystals arranged along the direction of muscle fibers (Fig.2 Aii and Aiii) caused by the slender and narrow interstitial space, which was in consistent with the mechanism that intermolecular channels induced orientation of hydroxyapatite crystals in mineralized collagen 63 . Differently, the regular structure of myotome disappeared due to the strong destructive effect of NaOH on the muscle fiber 75 (Fig.2 Ci), and calcium phosphate nucleated disordered on the organic matrix (Fig.2 Cii and Ciii). The unprecedented periodic stripe structure in GH group was further investigated. Streaky protrusions still could be observed on the surface of GH group after sectioning (Fig. 4A). What was more surprising, a significant mechanical difference between hydroxyapatite and muscle substrate was still exist even after resin embedding. In view of this, a modulus-displacement curve with a period of 1-2 microns was obtained through linear scanning (Fig. 4B). After 3D reconstruction, we clearly attained the modulus distribution in this region, and found the megapascal mechanical difference between HAP and resin-filled substrates, which could be easily recognized by cells, and then induce cell migration and differentiation 76-81 . Mapping the Raman absorption peak at 961cm -1 for PO 4 3- to evaluate the distribution of HAP in mineralized muscle fiber. And cyclic peak with a period of 1-2 microns was also exhibited, and the position of the peak corresponding to the bright band under light microscope (Fig. 4D). In addition, fluorescence staining was used to label amino and Ca, respectively, to reflect the relative positions of proteins and HAP: CY5.5-NHS for labelling proteins, and rhodamine B conjugated with alendronate sodium for labelling Ca, Fig. S9 depicted the synthesis process. Full of visual sense, red and green stripes in GH group were interleaved (Fig. 4E). Actually, red band after CY5.5 staining was a dark band under light microscope due to the overlap of proteins in myofibril. And HAP mineralization occurred in the bright zone, and the ordered streaks of HAP also could be clearly seen in TEM and EDS mapping (Fig. 4F). Unlike GH group, DI group and NaOH group showed a large overlap of red and green fluorescence (Fig. S10). Curiously, could this mineralized muscle with hierarchical ordered structure accelerate bone regeneration? Is it feasible to achieve the purpose of “Cutting muscle for bone regeneration”? Considering the similar anisotropy of mineralized muscle and femur, the middle of femur was chosen as the defect location. A cuboid defect (8 mm* 5 mm* 1.5mm) was constructed along the main axis of femur. Muscle extraction and bone defect implantation were depicted in Fig. S11. Unfortunately, due to the damage of mechanical properties of femur caused by large-sized defects and the compression by surrounding muscles, all rabbits in blank control group suffered from fractures or bone necrosis, and only one rabbit survived until 12 weeks. The corresponding CT reconstruction and histological staining results of blank control group were shown in Fig. S12. Scaffold implantation seemed to be conducive to timely closure of the defect site, and recover bone mechanical properties. After 12 weeks, all the implantation groups showed certain bone regeneration effects (Fig. S13, Movies S5-S8). Among them, GH group exhibited the best bone repair effect (Figure 5), although thickness at the defect center was slightly insufficient, the defect site was completely covered by new bone. And the bone repair rate (BV/TV) of GH group reached 85.28±5.04%, which was significantly higher than NaOH group (70.52±7.30%), DI group (73.38±9.94%) and pure muscle group (59.58±10.45%), and also significantly higher than current reported bone defect implantation materials (mostly 20-50%, Supplementary Table 1). Moreover, BV/TV of normal natural bone by CT scan with the same model was 87.41%. GH group also performed outstandingly in bone mineral density (BMD), trabecular bone thickness (Tb. Th), and trabecular bone separation (Tb. Sp) (Fig. S14). Thus, GH group demonstrated an awesome and reliable repair effect, and the bone mass at the defect site could almost be restored to the initial level of normal natural bone. The excellent bone repair ability of GH group was further proved by histological staining along the cross-section of defect center (Fig. 5E, 5F). Unlike the other groups, new bone completely covered the defect area, and defect center was not depressed under the compression of surrounding muscle in GH group. Micron-scale stripe structure in GH group was beneficial for MSCs migration (Fig. S15), which was also widely believed to facilitate the osteogenic differentiation of MSCs 82-84 . Although traditional scaffolds possessed certain ability to recruit stem cells and guide osteogenic differentiation, the pattern of gradual mineralization along the defect edge towards the center led to the inability of scaffold in central area to maintain support for a long time, especially as suffered from the pressure by surrounding muscles. Mineralized muscle with ordered stripes could recruit osteogenesis-related cells into defect center rapidly, and provide sufficient mechanical support to ensure the dynamic balance between scaffold degradation and bone regeneration. During regeneration, the defect area maintained good spatial integrity. As mineralized muscle partially degraded, along with new bone filled in (Fig. S16-S18). After 12 weeks, new bone in GH group was dense and mature (Fig. 5 Ei, Fi), and OCN staining also demonstrated its outstanding bone regeneration effect (Fig. 5Gi). However, there were still residual muscle fibers at defect site in NaOH group, which might be related to strong denaturation of muscle proteins caused by NaOH pretreatment. To evaluate the recovery of bone mechanical properties, hardness and elastic modulus at defect center were tested by nanoindentation technique. In GH group, the mechanical properties were recovered to approximately 90% of normal natural femur, both in terms of hardness and modulus (Fig. S19). For the other three groups, the mechanical properties of defect areas only could be restored by 30-50%. Since incomplete repair of defect center, resin embedding was used for samples fixing, the recovery data of bone mechanical properties may be somewhat higher than actual state in NaOH group, DI group, and muscle group, and also led to significant deviations. Conclusion Strategy, "Cutting muscle for bone regeneration", has been creatively proposed in this research. Autologous muscle as mineralization template, and anisotropic mineral stripes magically generated. Based on the unfolding of proteins induced by guanidine hydrochloride, this strategy fully utilized the complexation of troponin with Ca 2+ , as well as the spatial structure of interlaced arrangement of thick and thin filaments, ultimately achieved the striped mineral distribution within myofibrils. Periodic mineral stripes could recruit osteogenesis-related cells rapidly, and provide sufficient mechanical support to ensure the dynamic balance between scaffold degradation and bone regeneration. Surprisingly, the mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Excellent bone regeneration effect in vivo indicated this strategy was expected to solve the difficulty of insufficient autologous bone donors, and provided a new pathway for large-sized bone defects regeneration. Declarations Acknowledgments: This work was supported by National Natural Science Foundation of China (No. 51873187), Science Fund for Distinguished Young Scholars of Zhejiang Province (No.LR20E030004), National Basic Research Program of China (No. 2018YFC1004803), Research Project of Qizhen Innovation Concept Verification Center of Zhejiang University (No. GNYZ-2024010). Thanks for the help from Zhejiang University Instrument Platform. Author Contributions : X.L. conducted the experiments, analysed the experimental data and wrote the paper. Z.W. proposed the concept, supervised this research, and revised the manuscript. 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Mineralization process of muscle fibers for GH group. MovieS3DI.mp4 Movie S3. Mineralization process of muscle fibers for DI group. MovieS4.mp4 Movie S4. Expansion of muscle fiber after the introduction of Ca2+. MovieS5GH.mp4 Movie S5. CT reconstruction results of femoral defect site after 12 weeks (GH group). MovieS6NaOH.mp4 Movie S6. CT reconstruction results of femoral defect site after 12 weeks (NaOH group). MovieS7DI.mp4 Movie S7. CT reconstruction results of femoral defect site after 12 weeks (DI group). MovieS8Muscle.mp4 Movie S8. CT reconstruction results of femoral defect site after 12 weeks (Muscle group). Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7135552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":486348660,"identity":"3f7f7312-bc64-4112-8e1a-5fd4a763cbd8","order_by":0,"name":"Zhengke Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCAzYMjA1AmocELWmkazkMoYnSYnD87OGXP86ct2eekcD44G0bg7w5QS1n8tIsJG7cZmackcBsOLeNwXBnAwEtZgdyzAwMPtxmA2phk+ZtY0gwOEBIy/k3ZgYJH87xALWw/yZOy40c4wcHbhyQANnCTJQW+xtvzBgbziQbMPY8bJacc07CcAMhLZL9OcYffxyzszdsTz744U2ZjTxBW4CATQJEGjaAI1OCsHogYP4AIuWJUjsKRsEoGAUjEgAAhk5C9gofkZUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1474-1075","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Zhengke","middleName":"","lastName":"Wang","suffix":""},{"id":486348661,"identity":"98991b23-ffe8-4c16-afb9-393d8425b459","order_by":1,"name":"Xiaoyang Liu","email":"","orcid":"https://orcid.org/0000-0003-1424-1094","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-07-16 04:10:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7135552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7135552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86908984,"identity":"a6386f8f-1411-46b1-9869-1ba86df8bd27","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788880,"visible":true,"origin":"","legend":"\u003cp\u003eThe hierarchy of muscle fibers (A) and collagen fibers (B). The muscle hierarchy was inspired by muscle-tendon icon provided by Servier (https://smart.servier.com/). TEM image of collagen fiber in B adapted with permission from\u003csup\u003e65\u003c/sup\u003e, Copyright 2024, American Chemical Society.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/5b014f93a7de2b644d376077.png"},{"id":86909141,"identity":"000077d3-a9cf-48c9-aeb1-27bb8664ee69","added_by":"auto","created_at":"2025-07-17 04:47:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":488199,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of minerals in different pretreated muscle fibers: (A) DI group, (B) GH group, (C) NaOH group; i: TEM image of pretreated muscle fibers (non-mineralization), ii and iii: TEM image of mineralized muscle fibers at different magnification. The purple crystals in the scheme represented inorganic minerals.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/b77a6aae9fec1c1eb9994dca.jpeg"},{"id":86908987,"identity":"94a23a20-8385-48ec-a93e-3b584af0cad5","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":490234,"visible":true,"origin":"","legend":"\u003cp\u003eC1s, O1s, and P2p spectra of high-resolution X-ray photoelectron spectroscopy: (A) pure muscle, (B) GH group. SAXS patterns: (C) muscle pretreated by guanidine hydrochloride (without mineralization), (D) GH group. (E) XRD patterns of muscle and GH group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/79e8359e7fe571e5294e6b01.png"},{"id":86908993,"identity":"346a9283-1f19-49ed-a5d6-df56c3c34868","added_by":"auto","created_at":"2025-07-17 04:39:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1448109,"visible":true,"origin":"","legend":"\u003cp\u003eOrdered streaky distribution of hydroxyapatite in GH group: (A) AFM mapping of GH group slice. (B) Modulus mapping of GH slice, and Young's modulus-displacement curve marked along the white line. (C) 3D image of Young's modulus of GH group slice obtained by AFM. (D) Raman spectra of GH group in aqueous environment, and the peak area-displacement curve at 961cm\u003csup\u003e-1 \u003c/sup\u003e(PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e) obtained by drawing lines along the marked position within the dotted box. (E) Fluorescent staining for GH group, the protein was red stained by CY5.5- NHS, while Ca\u003csup\u003e2+\u003c/sup\u003e was marked green by rhodamine B modified with sodium alendronate. (F) High angle ring dark field image of GH group (DF), and corresponding EDS mappings.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/288129b277418b60f4c3381d.png"},{"id":86908988,"identity":"0e620b68-d88a-46ec-ba66-108a730bf149","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":879916,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of bone regeneration effect. Axial, sagittal, and 3D micro-CT images of rabbit femur regeneration after implantation for 12 weeks: (A) GH, (B) NaOH, (C) DI, (D) pure muscle. The dotted orange box indicated the defect location. H\u0026amp;E (E) and Masson (F) staining of each group after 12 weeks. Black asterisk represented new bone, while red arrow indicated the residual scaffold. G: Immuno-histological characterizations for OCN in femoral defect model. In Figure E and F, initial defect areas were marked with two vertical dotted lines; and i, ii, iiiand ivrepresented GH group, NaOH group, DI group, and pure muscle group, respectively. Scale bars: 2μm.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/bd72b80c1bfec2eb0864a9e2.jpeg"},{"id":88282925,"identity":"eb68beab-b1b5-4920-9221-3728c3be1c64","added_by":"auto","created_at":"2025-08-04 21:13:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4528286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/c6160141-0f5e-482b-93af-18fd5f0031bc.pdf"},{"id":86909142,"identity":"8ec2e0d2-28b8-4a18-aa42-c86f8e7b8b4d","added_by":"auto","created_at":"2025-07-17 04:47:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2541615,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"SupportingInformation20250626.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/ebacbcf3e4fa27a2f26b479b.pdf"},{"id":86908986,"identity":"5ea34999-d54b-485b-a897-0786828a412e","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1884993,"visible":true,"origin":"","legend":"\u003cp\u003eStriped mineralized muscle\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/34cfc6efa97205800506b178.jpg"},{"id":86908989,"identity":"5d40a336-ec64-4b17-99fc-ab8c3da1ddbb","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5200360,"visible":true,"origin":"","legend":"\u003cp\u003eMovie S1. Mineralization process of muscle fibers for NaOH group.\u003c/p\u003e","description":"","filename":"MovieS1NaOH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/d235247ce831fee9462a9a20.mp4"},{"id":86908990,"identity":"be871338-ac94-4dd3-a3a2-45fa7e7a3cf7","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5119052,"visible":true,"origin":"","legend":"\u003cp\u003eMovie S2. Mineralization process of muscle fibers for GH group.\u003c/p\u003e","description":"","filename":"MovieS2GH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/1d4fe7c775f8cb4e31551b88.mp4"},{"id":86908991,"identity":"2c5f7a8a-3acc-4aa3-8ffe-541705ab1c78","added_by":"auto","created_at":"2025-07-17 04:39:46","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4509341,"visible":true,"origin":"","legend":"\u003cp\u003eMovie S3. Mineralization process of muscle fibers for DI group.\u003c/p\u003e","description":"","filename":"MovieS3DI.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/8706f72e8a46c8765a8c4c54.mp4"},{"id":86909143,"identity":"9e47bb28-ff44-4a68-9441-33c178d2a553","added_by":"auto","created_at":"2025-07-17 04:47:47","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4989756,"visible":true,"origin":"","legend":"Movie S4. Expansion of muscle fiber after the introduction of Ca2+.","description":"","filename":"MovieS4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/6eccc743edd8e8b235b68cc4.mp4"},{"id":86908995,"identity":"65647996-cb48-4f2d-bd6c-d8ab348f6572","added_by":"auto","created_at":"2025-07-17 04:39:47","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5751717,"visible":true,"origin":"","legend":"Movie S5. CT reconstruction results of femoral defect site after 12 weeks (GH group).","description":"","filename":"MovieS5GH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/96c4e2b13e1439a4ec390bcc.mp4"},{"id":86908997,"identity":"b8ead77f-6bb6-48b6-94cd-587cfbf4306c","added_by":"auto","created_at":"2025-07-17 04:39:47","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6007714,"visible":true,"origin":"","legend":"Movie S6. CT reconstruction results of femoral defect site after 12 weeks (NaOH group).","description":"","filename":"MovieS6NaOH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/3566ab305bbf541fb043abfd.mp4"},{"id":86908998,"identity":"b2e682fe-db4b-4461-bf82-1a1d93133d93","added_by":"auto","created_at":"2025-07-17 04:39:47","extension":"mp4","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5636716,"visible":true,"origin":"","legend":"Movie S7. CT reconstruction results of femoral defect site after 12 weeks (DI group).","description":"","filename":"MovieS7DI.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/0a0673446755b05f2d778f89.mp4"},{"id":86908994,"identity":"f02757cb-03ad-49cc-9fdf-2d2f3445e362","added_by":"auto","created_at":"2025-07-17 04:39:47","extension":"mp4","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":5667587,"visible":true,"origin":"","legend":"Movie S8. CT reconstruction results of femoral defect site after 12 weeks (Muscle group).","description":"","filename":"MovieS8Muscle.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7135552/v1/f7f538cbfdf8869136882bda.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cutting muscle for bone regeneration (割肉修骨)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBone, a highly mineralized tissue with complex hierarchical structure, is responsible for bearing loads, protecting fragile organs, maintaining hematopoietic and mineral balance\u003csup\u003e1-3\u003c/sup\u003e. Through the regulation of osteoblasts and osteoclasts, bone maintains a dynamic balance of bone resorption and bone remodeling. Thus, small-sized bone defects can self-heal through continuous bone renewal without external intervention as long as no dislocation occurs\u003csup\u003e4\u003c/sup\u003e. However, clinically, large bone defect that exceed critical size due to disease or trauma is still a major challenge. According to statistics, more than 20 million people worldwide suffer from bone defects every year, and about 60% of them need bone grafting to promote bone repair\u003csup\u003e5\u003c/sup\u003e. Although autograft still serves as the\u0026nbsp;gold standard for bone defect repair, it also faces the disadvantages of limited donors\u003csup\u003e6,7\u003c/sup\u003e. Therefore, numerous researches have been devoted to developing biodegradable scaffolds for the repair of large-size bone defects.\u003c/p\u003e\n\u003cp\u003eTraditionally, Ca-P mineral scaffolds appear to be preferred for their similar composition with bone and excellent osteoinductivity\u003csup\u003e8-11\u003c/sup\u003e. Of course, many researchers found that some inorganic ions such as Cu\u003csup\u003e12,13\u003c/sup\u003e, Zn\u003csup\u003e12,14\u003c/sup\u003e, Mg\u003csup\u003e15-17\u003c/sup\u003e, Se\u003csup\u003e18\u003c/sup\u003e, Sr\u003csup\u003e14\u003c/sup\u003e, Ce\u003csup\u003e19\u003c/sup\u003e, Si\u003csup\u003e20-22\u003c/sup\u003e etc. could also accelerate bone regeneration. Further, not limited to the change of components, the design of anisotropic structure and surface topography is also one of the research hotspots\u003csup\u003e23-32\u003c/sup\u003e. To date, researchers have tried various strategies including hyperthermia\u003csup\u003e33-37\u003c/sup\u003e, electrotherapy\u003csup\u003e38\u003c/sup\u003e, magnetic therapy\u003csup\u003e39,40\u003c/sup\u003e, immunomodulation\u003csup\u003e41-48\u003c/sup\u003e, cells\u003csup\u003e20,49,50\u003c/sup\u003e or growth factors\u003csup\u003e51-59\u003c/sup\u003e loading to promote the repair of large bone defects. The concept of BV/TV (bone volume/tissue volume) also was introduced\u0026nbsp;to quantitatively assess the effect of bone regeneration. Regrettably, most of these well-designed scaffolds only achieve 20-50% repair rate after implantation for 8 or 12 weeks (Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eFacing the dilemma of poor bone repair effect, drawing inspiration from nature seems to bring a new way. Researches are dedicated to studying the structure of natural bones from micro to macro level\u003csup\u003e60\u003c/sup\u003e. On the basis of previous researches, Roland Kr\u0026ouml;ger et al. further subdivided the\u0026nbsp;skeletal structure into 12 hierarchies\u003csup\u003e61\u003c/sup\u003e. Given the important role of collagen as organic templates in bone formation, many endeavors to reveal the intrafibrillar or extrafibrillar mineralization process have been made\u003csup\u003e62-65\u003c/sup\u003e. In fact, these studies mainly focused on the mechanism of collagen fiber mineralization in vitro. However, building complex hierarchies of collagen in vitro, similar to that in natural bone, is still difficult to achieve. Interestingly, as shown in Fig.1, the hierarchical structure of skeletal muscle (myofibril - muscle fiber - muscle fascicle - skeletal muscle) is highly similar to that of collagen (collagen microfiber - collagen fiber - collagen fiber bundle) in bone\u003csup\u003e66,67\u003c/sup\u003e. It is well known that collagen fibers exhibit a characteristic 67nm streak, due to the cross-arrangement of protocollagen. Correspondingly, the staggered arrangements of thick filaments and thin filaments lead to the light and dark stripes (2\u0026nbsp;\u0026mu;m). Bionics from natural bone, firstly create an organic template (hierarchical collagen fibers), then mineralize to form hydroxyapatite with ordered structures. Interestingly, is it feasible to select hierarchical muscle fibers as a mineralization template instead of collagen fibers, and then achieve excellent bone regeneration?\u003c/p\u003e\n\u003cp\u003eHerein, \u0026ldquo;cutting muscle for bone regeneration\u0026rdquo; is validated in this research. Surprisingly, after skeletal muscle treated with guanidine hydrochloride, mineralized hydroxyapatite is striped arranged, and perpendicular to the direction of the myofibril. More importantly, this mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Compared with bone, muscle is easy to regenerate, and bone defect regeneration can be achieved by mineralized muscle, which can solve the difficulty of insufficient autologous bone donors. Replacing collagen fibers with muscle fibers, studying the mineralization mechanism of hydroxyapatite, and preparing biomimetic materials with hierarchical ordered structure, similar to natural bone. \u0026quot;Cutting muscle for bone regeneration\u0026quot; is a good combination of biomimetic materials science and practical applications, which provides a new approach for orthopedic clinical practice.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eFresh pork tenderloin was cut into slices, and soaked in acetone overnight to remove lipids. After degreasing, the acetone in muscle was exchanged by deionized water. Then, muscle slices were treated with deionized water (DI), guanidine hydrochloride solution (GH, 2M) and sodium hydroxide solution (NaOH, 5wt%) for 3h, respectively, and named DI group, GH group, and NaOH group. Cyclic mineralization of muscles was then taken by immersion in calcium chloride solution, sodium dihydrogen phosphate solution, and alkaline water in sequence, and terminated as the mineral content reached around 20wt%, lyophilized.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePretreatment and mineralization processes of individual muscle fibers were recorded in Movies S1-S3 (S1 for NaOH group, S2 for GH group, and S3 for DI group). Compared with the initial state, introduction of guanidine hydrochloride made muscle fibers swollen and loose, while sodium hydroxide had a stronger effect. After immersion in Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003esolution, the muscle fiber exhibited obvious expansion along the cross-section (Movie S4), attributed to the complexation of Ca\u003csup\u003e2+\u003c/sup\u003e and troponin. After introducing calcium ions and phosphate ions successively, the muscle fibers were immersed in alkaline water, and expanded first and then contracted. Contraction in collagen fibrils by mineralization was discovered by Peter Fratzl\u003csup\u003e68\u003c/sup\u003e, and this is the same reason for the contraction of muscle fibers during mineralization.\u003c/p\u003e\n\u003cp\u003eMineral in muscle existed in the form of hydroxyapatite regardless of how it was pre-treated (Fig. S1 and S2), and (002), (121) crystal planes of HAP were also observed on the selected electron diffraction pattern (Fig. S3). Indeed, hydroxyapatite in muscle was polycrystalline with low crystallinity. Ca and P were uniformly distributed in mineralized muscle fibers (Cross-section, Fig. S4). At the mesoscopic scale, there was no obvious difference in CT scans of the mineralized muscles with three different pretreatment methods (Fig. S5). However, at a microscopic scale, mineral distribution emerged obvious differences in DI, GH, and NaOH groups (Fig. 2). There were distinctive light and dark stripes in pure muscle fibers, sarcomere and edge were clear (Fig. 2Ai). Correspondingly, the mineral phases with long acicular crystals were preferentially arranged along the direction of muscle fibers in DI group (Fig. 2Aii), and distributed in the space between myofibril (Fig. S6C). While muscle pretreated with guanidine hydrochloride, myofibril became fluffier, and myotome remained clear, but the edge became frizz (Fig. 2Bi). Surprisingly, the mineral phases appeared inside the myofibril (Fig. S6A), and showed a streaky distribution perpendicular to the direction of muscle fibers, with mineralized streaks spaced 1-2 microns apart (Fig. 2Bii). Small-angle diffraction pattern also indicated ordered stripe mineralization for GH group (Fig. 3D), which was not seen in pure muscle (Fig. 3C), DI group (Fig. S7A) and NaOH group (Fig. S7B).\u0026nbsp;Compared with guanidine hydrochloride, a strong alkaline environment seemed to have significantly stronger disaggregation effect on muscle protein \u003csup\u003e69,70\u003c/sup\u003e. Myofibril structure was damaged by alkali treatment (Fig. 2Ci): myofibrils seemed to adhere together, and myotome structure was difficult to distinguish. Therefore, no matter from which angle, the mineral phases exhibited disorder distribution in NaOH group (Fig. 2Cii and Fig. S6B).\u003c/p\u003e\n\u003cp\u003eInterestingly, why minerals were orderly arranged in GH group? Muscle protein was unfolded by guanidine hydrochloride \u003csup\u003e71,72\u003c/sup\u003e, and C-N/C-O signals after mineralization shifted from 286.06 eV to 285.35 eV, compared with pure muscle (Fig. 3). It was worth noting that the P2p signals in pure muscle, 134.02 eV and 133.07 eV, were derived from the remaining ATP-like substance, rather than calcium phosphate minerals. While P2p\u003csub\u003e1/2\u003c/sub\u003e and P2\u003csub\u003e3/2\u003c/sub\u003e located at 132.56 and 131.61 eV, respectively, for GH group. And O1s with a signal peak at 534.95eV for GH group, was not seen in any other group, and also accompanied by a decrease in the intensity of O=C peak (Fig. 3, Fig.S8). Considering the strong complexation of troponin to Ca\u003csup\u003e2+\u003c/sup\u003e during muscle contraction\u003csup\u003e73,74\u003c/sup\u003e and the striate distribution of minerals at the bright region of sarcomere in GH group (Fig.2 Bii), it was reasonable to speculate that Ca\u003csup\u003e2+\u003c/sup\u003e strongly ligated with carboxyl site of troponin during mineralization, and induced mineral growth as a nucleus. Guanidine hydrochloride changed the conformation of muscle protein\u003csup\u003e72\u003c/sup\u003e, which made the myotome structure less dense (Fig.2 Bi), and exogenous Ca\u003csup\u003e2+\u003c/sup\u003e could enter the interior of myotome and complex with troponin. Differently, the dense sarcomere structure in DI group prevented the traverse of Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e(Fig.2 Ai), and calcium phosphate mineral was forced to nucleate and grow in the interstitial space of myofibril. Thus, HAP long crystals arranged along the direction of muscle fibers (Fig.2 Aii and Aiii) caused by the slender and narrow interstitial space, which was in consistent with the mechanism that intermolecular channels induced orientation of hydroxyapatite crystals in mineralized collagen\u003csup\u003e63\u003c/sup\u003e. Differently, the regular structure of myotome disappeared due to the strong destructive effect of NaOH on the muscle fiber \u003csup\u003e75\u003c/sup\u003e (Fig.2 Ci), and calcium phosphate nucleated disordered on the organic matrix (Fig.2 Cii and Ciii).\u003c/p\u003e\n\u003cp\u003eThe unprecedented periodic stripe structure in GH group was further investigated. Streaky protrusions still could be observed on the surface of GH group after sectioning (Fig. 4A). What was more surprising, a significant mechanical difference between hydroxyapatite and muscle substrate was still exist even after resin embedding. In view of this, a modulus-displacement curve with a period of 1-2 microns was obtained through linear scanning (Fig. 4B). After 3D reconstruction, we clearly attained the modulus distribution in this region, and found the megapascal mechanical difference between HAP and resin-filled substrates, which could be easily recognized by cells, and then induce cell migration and differentiation\u003csup\u003e76-81\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMapping the Raman absorption peak at 961cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003efor PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u0026nbsp;\u003c/sup\u003eto evaluate the distribution of HAP in mineralized muscle fiber. And cyclic peak with a period of 1-2 microns was also exhibited, and the position of the peak corresponding to the bright band under light microscope (Fig. 4D). In addition, fluorescence staining was used to label amino and Ca, respectively, to reflect the relative positions of proteins and HAP: CY5.5-NHS for labelling proteins, and rhodamine B conjugated with alendronate sodium for labelling Ca, Fig. S9 depicted the synthesis process. Full of visual sense, red and green stripes in GH group were interleaved (Fig. 4E). Actually, red band after CY5.5 staining was a dark band under light microscope due to the overlap of proteins in myofibril. And HAP mineralization occurred in the bright zone, and the ordered streaks of HAP also could be clearly seen in TEM and EDS mapping (Fig. 4F). Unlike GH group, DI group and NaOH group showed a large overlap of red and green fluorescence (Fig. S10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCuriously, could this mineralized muscle with hierarchical ordered structure accelerate bone regeneration? Is it feasible to achieve the purpose of\u0026nbsp;\u0026ldquo;Cutting muscle for bone regeneration\u0026rdquo;? Considering the similar anisotropy of mineralized muscle and femur, the middle of femur was chosen as the defect location. A cuboid defect (8 mm* 5 mm* 1.5mm) was constructed along the main axis of femur. Muscle extraction and bone defect implantation were depicted in Fig. S11. Unfortunately, due to the damage of mechanical properties of femur caused by large-sized defects and the compression by surrounding muscles, all rabbits in blank control group suffered from fractures or bone necrosis, and only one rabbit survived until 12 weeks. The corresponding CT reconstruction and histological staining results of blank control group were shown in Fig. S12. Scaffold implantation\u0026nbsp;seemed to be conducive to timely closure of the defect site, and recover bone mechanical properties. After 12 weeks, all the implantation groups showed certain bone regeneration effects (Fig. S13, Movies S5-S8).\u0026nbsp;Among them, GH group exhibited the best bone repair effect (Figure 5), although thickness at the defect center was slightly insufficient, the defect site was completely covered by new bone. And the bone repair rate (BV/TV) of GH group reached 85.28\u0026plusmn;5.04%, which was significantly higher than NaOH group (70.52\u0026plusmn;7.30%), DI group (73.38\u0026plusmn;9.94%) and pure muscle group (59.58\u0026plusmn;10.45%), and also significantly higher than current reported bone defect implantation materials (mostly 20-50%, Supplementary Table 1). Moreover, BV/TV of normal natural bone by CT scan with the same model was 87.41%. GH group also performed outstandingly in bone mineral density (BMD), trabecular bone thickness (Tb. Th), and trabecular bone separation (Tb. Sp) (Fig. S14). Thus, GH group demonstrated an awesome and reliable repair effect, and the bone mass at the defect site could almost be restored to the initial level of normal natural bone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe excellent bone repair ability of GH group was further proved by histological staining along the cross-section of defect center (Fig. 5E, 5F). Unlike the other groups, new bone completely covered the defect area, and defect center was not depressed under the compression of surrounding muscle in GH group. Micron-scale stripe structure in GH group was beneficial for MSCs migration (Fig. S15), which was also widely believed to facilitate the osteogenic differentiation of MSCs\u003csup\u003e82-84\u003c/sup\u003e. Although traditional scaffolds possessed certain ability to recruit stem cells and guide osteogenic differentiation, the pattern of gradual mineralization along the defect edge towards the center led to the inability of scaffold in central area to maintain support for a long time, especially as suffered from the pressure by surrounding muscles. Mineralized muscle with ordered stripes could recruit osteogenesis-related cells into defect center rapidly, and provide sufficient mechanical support to ensure the dynamic balance between scaffold degradation and bone regeneration. During regeneration, the defect area maintained good spatial integrity. As mineralized muscle partially degraded, along with new bone filled in (Fig. S16-S18). After 12 weeks, new bone in GH group was dense and mature (Fig. 5 Ei, Fi), and OCN staining also demonstrated its outstanding bone regeneration effect (Fig. 5Gi). However, there were still residual muscle fibers at defect site in NaOH group, which might be related to strong denaturation of muscle proteins caused by NaOH pretreatment.\u003c/p\u003e\n\u003cp\u003eTo evaluate the recovery of bone mechanical properties, hardness and elastic modulus at defect center were tested by nanoindentation technique. In GH group, the mechanical properties were recovered to approximately 90% of normal natural femur, both in terms of hardness and modulus (Fig. S19). For the other three groups, the mechanical properties of defect areas only could be restored by 30-50%. Since incomplete repair of defect center, resin embedding was used for samples fixing, the recovery data of bone mechanical properties may be somewhat higher than actual state in NaOH group, DI group, and muscle group, and also led to significant deviations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eStrategy, \u0026quot;Cutting muscle for bone regeneration\u0026quot;, has been creatively proposed in this research. Autologous muscle as mineralization template, and anisotropic mineral stripes magically generated. Based on the unfolding of proteins induced by guanidine hydrochloride, this strategy fully utilized the complexation of troponin with Ca\u003csup\u003e2+\u003c/sup\u003e, as well as the spatial structure of interlaced arrangement of thick and thin filaments, ultimately achieved the striped mineral distribution within myofibrils. Periodic mineral stripes could recruit osteogenesis-related cells rapidly, and provide sufficient mechanical support to ensure the dynamic balance between scaffold degradation and bone regeneration. Surprisingly, the mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Excellent bone regeneration effect \u003cem\u003ein vivo\u003c/em\u003e indicated this strategy was expected to solve the difficulty of insufficient autologous bone donors, and provided a new pathway for large-sized bone defects regeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (No. 51873187), Science Fund for Distinguished Young Scholars of Zhejiang Province (No.LR20E030004), National Basic Research Program of China (No. 2018YFC1004803), Research Project of Qizhen Innovation Concept Verification Center of Zhejiang University (No. GNYZ-2024010). Thanks for the help from Zhejiang University Instrument Platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.L. conducted the experiments, analysed the experimental data and wrote the paper. Z.W. proposed the concept, supervised this research, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eeclaration of interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, X.D., Wang, T., Zhang, Z.Y., Liu, H.Q., Li, L.F., Wang, A.C., Ouyang, J., Xie, T., Zhang, L.Q., Xue, J.J., and Tao, W. (2023). 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Bioactive Materials \u003cem\u003e27\u003c/em\u003e, 181-199. https://doi.org/10.1016/j.bioactmat.2023.03.024.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hierarchical structure, muscle, biomineralization, bone regeneration","lastPublishedDoi":"10.21203/rs.3.rs-7135552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7135552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"As gold standard for large-sized bone defects regeneration, autologous bone transplantation has always faced the difficulty of insufficient donors. Moreover, most of the well-designed biomaterials could only achieve 20-50% repair rate. Herein, we innovatively proposed the strategy of \"Cutting muscle for bone regeneration\". Considering the hierarchical structure of muscle was similar to collagen in bone, we prepared hierarchical mineralized muscle for bone regeneration. Based on the unfolding of proteins induced by guanidine hydrochloride, this strategy fully utilized the complexation of troponin with Ca2+, as well as the spatial structure of interlaced arrangement of thick and thin filaments, ultimately achieved the striped mineral distribution within myofibrils. Surprisingly, the mineralized muscle greatly promoted the bone regeneration, and basically achieved complete repair after 3 months. Compared with bone, muscle is easy to regenerate, and bone defect regeneration can be achieved by mineralized muscle, which can solve the difficulty of insufficient autologous bone donors.","manuscriptTitle":"Cutting muscle for bone regeneration (割肉修骨)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 04:39:41","doi":"10.21203/rs.3.rs-7135552/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb55646d-7b42-4fc2-8371-c68b129d6c52","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51631223,"name":"Physical sciences/Materials science/Biomaterials/Bioinspired materials"},{"id":51631224,"name":"Physical sciences/Materials science/Biomaterials/Biomineralization"},{"id":51631225,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2025-08-05T02:10:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 04:39:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7135552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7135552","identity":"rs-7135552","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}