Neonatal Enthesis Healing Involves Noninflammatory Acellular Scar Formation through Extracellular Matrix Secretion by Resident Cells
2022; Elsevier BV; Volume: 192; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2022.05.008
ISSN1525-2191
AutoresRon Carmel Vinestock, Neta Felsenthal, Eran Assaraf, Eldad Katz, Sarah Rubin, Lia Heinemann‐Yerushalmi, Sharon Krief, Nili Dezorella, Smadar Levin‐Zaidman, Michael Tsoory, Stavros Thomopoulos, Elazar Zelzer,
Tópico(s)Connective tissue disorders research
ResumoWound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries. Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries. Wound healing is a critical and complex process that restores structure and function by replacing damaged tissue. In adult animals, this process comprises a sequential cascade of overlapping events, including bleeding and activation of the coagulation system, recruitment of inflammatory cells, fibroblast migration, collagen synthesis, angiogenesis, and remodeling of the injury site.1Darby I.A. Desmoulière A. Scar formation: cellular mechanisms. Textbook on Scar Management.in: Téot L. Mustoe T.A. Middelkoop E. Gauglitz G.G. Springer International Publishing, Cham, Switzerland2020: 19-26Google Scholar,2Stroncek J.D. Reichert W.M. Overview of wound healing in different tissue types.Indwelling Neural Implants: Strategies for Contending with the In Vivo Environment. CRC Press, Boca Raton, FL2007: 3-38Crossref Google Scholar In the musculoskeletal system, however, tissues differ in their ability to heal injuries.3Shen W. Ferretti M. Manley M. Fu F. Musculoskeletal fundamentals: form, function, and a survey of healing strategies.Musculoskeletal Tissue Regeneration: Biological Materials and Methods. 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An ultrastructural study of chondroptosis: programmed cell death in degenerative intervertebral discs in vivo.J Anat. 2017; 231: 129-139Crossref PubMed Scopus (5) Google Scholar In full-thickness injuries, the damage is deeper and reaches into the subchondral bone, thereby generating a pathway into the vascularized bone marrow. This enables access for the recruitment of immune cells, eventually leading to fibrocartilage formation and ECM deposition in the wound area. As the newly formed fibrocartilage is weaker than the original tissue, the cartilage undergoes gradual degradation, which leads to loss of mechanical properties.22Matsiko A. Levingstone T.J. O'Brien F.J. Advanced strategies for articular cartilage defect repair.Materials (Basel). 2013; 6: 637-668Crossref PubMed Scopus (86) Google Scholar, 23Hunziker E.B. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable?.Osteoarthr Cartil. 1999; 7: 15-28Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 24Newman A.P. Articular cartilage repair.Am J Sports Med. 1998; 26: 309-324Crossref PubMed Scopus (371) Google Scholar Another component of the musculoskeletal system is the enthesis, a fibrocartilaginous tissue that bridges tendon and bone. This unique specialized connective tissue attaches the two distinct tissues by forming a gradient of cellular and extracellular features along its length.25Doschak M.R. Zernicke R.F. Structure, function and adaptation of bone-tendon and bone-ligament complexes.J Musculoskelet Neuronal Interact. 2005; 5: 35-40PubMed Google Scholar,26Zelzer E. Blitz E. Killian M.L. Thomopoulos S. Tendon-to-bone attachment: from development to maturity.Birth Defects Res C Embryo Today. 2014; 102: 101-112Crossref PubMed Scopus (97) Google Scholar Adult enthesis repair commonly results in permanent damage because of failure to restore its structure. The resulting mechanically insufficient attachment limits joint function and is prone to retears.27Derwin K.A. Galatz L.M. Ratcliffe A. Thomopoulos S. Enthesis repair: challenges and opportunities for effective tendon-to-bone healing.J Bone Joint Surg Am. 2018; 100: e109Crossref PubMed Scopus (45) Google Scholar However, the outcome may depend on the severity and type of injury. Comparison of the mechanical outcomes of rotator cuff injuries in adult mice suggest that repair is more successful after partial injuries, including reduced scar formation and recovery of gait.28Moser H.L. Doe A.P. Meier K. Garnier S. Laudier D. Akiyama H. Zumstein M.A. Galatz L.M. Huang A.H. Genetic lineage tracing of targeted cell populations during enthesis healing.J Orthop Res. 2018; 36: 3275-3284Crossref PubMed Scopus (17) Google Scholar Furthermore, rotator cuff entheses of neonatal mice display some regenerative capacity.29Schwartz A.G. Galatz L.M. Thomopoulos S. Enthesis regeneration: a role for Gli1+ progenitor cells.Development. 2017; 144: 1159-1164PubMed Google Scholar The difference in healing potential between adult and neonatal mice raises the possibility that the healing capacity of the enthesis is switched off early after birth. Moreover, given the complexity of the enthesis structure, it is unclear whether the healing process, if exists, follows the same course in all enthesis zones. To address these questions, needle-punch injury was induced to neonatal Achilles enthesis, an injury model that minimizes damage to surrounding tissues or penetration into the bone marrow space. Although no inflammation or angiogenesis was observed, temporal analyses identified the formation of an acellular domain and ECM plug at the injury site. The acellular domain was composed of COL1A1 at the tendon end and collagen type II (COL2A1) at the cartilage end, suggesting that this domain is formed locally by the resident enthesis cells. Immunostaining, gene expression analyses, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy (TEM) revealed that cells at the injury site undergo ER stress and autophagy, and suggested that the observed cellular loss was a result of chondroptosis-like cell death. Gait recovery suggested that, despite the loss of tissue structure, the healing process effectively restored joint function. Together, these findings reveal a novel mechanism in neonatal mice whereby extensive secretion of ECM by resident cells at the injury site drives enthesis healing and restores its function. All experiments involving mice were approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science (Rehovot, Israel). Histology was performed on C57BL6 wild-type mice. Green fluorescent protein (GFP)–LC3#53 mice were kindly provided by Prof. Zvulun Elazar (Weizmann Institute of Science). Postnatal day 7 neonatal mice were anesthetized by lidocaine (0.03 mg, intraperitoneally). A small incision was made through the skin to expose the Achilles enthesis and was needle punched using a 32-gauge needle using a sterile approach. The skin was then sutured with nylon 5/0 monofilament. The left limb was used as a control. After injury, the animals returned to full cage activity. Sex distribution was equal. For histology, postnatal mice were harvested at various ages, dissected, and fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) at 4°C overnight. After fixation, tissues were dehydrated to 70% ethanol and embedded in paraffin. Pup and adult tissues were decalcified using 0.5 mol/L EDTA (pH 7.4) before dehydration. The embedded tissues were cut to generate sections (7 μm thick) and mounted onto slides. Hematoxylin and eosin and Safranin O stains were performed following standard protocols. TUNEL assay was performed using In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), according to the manufacturer's protocol. For Von Kossa and toluidine blue (pH 6.0) staining, fixed calcified tissues were embedded in optimal cutting temperature compound. Cryosections (10 μm thick) were prepared using the Kawamoto film method,30Kawamoto T. Kawamoto K. Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot's film method (2012).Methods Mol Biol. 2014; 1130: 149-164Crossref PubMed Scopus (125) Google Scholar and staining was performed using standard protocols.31Bemenderfer T.B. Harris J.S. Condon K.W. Li J. Kacena M.A. Processing and sectioning undecalcified murine bone specimens.Methods Mol Biol. 2021; 2230: 231-257Crossref PubMed Scopus (2) Google Scholar In short, postnatal mice were harvested at 5 months after injury, dissected, and fixed in 4% PFA/PBS at 4°C overnight. After fixation, tissues were transferred to 30% sucrose overnight, then embedded in OCT and sectioned by cryostat at a thickness of 10 μm. Slides were incubated in 1% silver nitrate solution for 2.5 minutes in UV table, then rinsed three times in distilled water. Unreacted silver was removed by 5-minute incubation in 5% sodium thiosulfate, then rinsed in water. Then, sections were counterstained with toluidine blue. Last, slides were mounted with Entellan (Sigma-Aldrich, St. Louis, MO; 1079600500). For immunohistochemistry on paraffin sections, animals were harvested at various ages, dissected, and fixed in 4% PFA/PBS at 4°C overnight. After fixation, tissues were decalcified using 0.5 mol/L EDTA (pH 7.4), washed thoroughly with water, dehydrated to 70% ethanol, and embedded in paraffin. The embedded tissues were cut to generate sections (7 μm thick) and mounted onto slides. Antigen retrieval for anti-collagen types I, II, and III antibodies was performed using 1.8 μg proteinase K (P9290; Sigma-Aldrich) in 200 mL PBS for 10 minutes. Antigen retrieval for anti-GFP, F4/80, and cleaved caspase-3 antibodies was performed in 10 mmol/L sodium citrate buffer (pH 6.0) cooked in 80°C for 15 minutes in a hot tub. Then, sections were washed twice in PBS, and endogenous peroxidase was quenched using 3% H2O2 in PBS. Non-specific binding was blocked using 7% horse serum and 1% bovine serum albumin dissolved in PBS–Tween 20 (PBST) for 1 hour. Then, sections were incubated with rabbit anti-collagen I antibody (1:100; number NB600-408; Novus Biologicals, Littleton, CO), mouse anti-collagen II antibody (1:50; II-II6B3; Developmental Studies Hybridoma Bank, Iowa City, IA), rabbit anti-collagen III (1:100; ab7778; Abcam, Cambridge, UK), goat anti-GFP (biotin) antibody (1:100; ab6658; Abcam), rabbit anti-cleaved caspase-3 (Asp175) antibody (1:200; number 9664s; Cell Signaling Technology, Danvers, MA), or rat anti-F4/80 antibody (1:50; ab6640; Abcam) overnight at room temperature. The next day, sections were washed twice in PBST and incubated with biotin anti-rabbit (1:100; Jackson ImmunoResearch, West Grove, PA), or biotin anti-rat (1:100; Jackson ImmunoResearch), for 1 hour at room temperature. Then, after two washes of PBST, slides were incubated with streptavidin-Cy2 or streptavidin-Cy3 (1:100; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-mouse (1:100; Jackson ImmunoResearch). Occasionally, slides were counterstained using DAPI. Then, slides were mounted with Shandon Immu-mount (number 9990402; Thermo Fisher Scientific, Waltham, MA). For immunohistochemistry on cryosections, animals were harvested at various ages, dissected, and fixed in 4% PFA/PBS at 4°C overnight. Then, tissues were decalcified using 0.5 mol/L EDTA (pH 7.4) and transferred to 30% sucrose overnight, embedded in OCT, and sectioned by cryostat at a thickness of 10 μm. Cryosections were dried and post-fixed for 20 minutes in acetone at −20°C. Then, sections were permeabilized with 0.2% Triton/PBS. To block non-specific binding of Ig, sections were incubated with 7% goat serum in PBS. Cryosections were then incubated overnight at 4°C with primary antibody rat anti-mouse CD31 (PMG550274; 1:50; BD Pharmingen, San Diego, CA). The next day, sections were washed in PBS and incubated with biotin anti-rabbit (1:100; Jackson ImmunoResearch). Then, slides were incubated with streptavidin-Cy3 (1:100; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-rabbit (1:100; Jackson ImmunoResearch). Occasionally, slides were counterstained using DAPI. Then, slides were mounted with Shandon Immu-mount. Animals were harvested at 1 and 3 days and 5 months after injury, dissected, and fixed with 3% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L cacodylate buffer containing 5 mmol/L CaCl2 (pH 7.4) overnight. The tissue was decalcified using 0.5 mol/L EDTA (pH 7.4) for 96 hours. Vibrotome sections (200 μm thick) were prepared (VT1000 S; Leica, Wetzlar, Germany), and tissue was postfixed in 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate trihydrate and potassium dichromate in 0.1 mol/L cacodylate (1 hour), stained with 2% uranyl acetate in water (1 hour), dehydrated in graded ethanol solutions, and embedded in Agar 100 epoxy resin (Agar Scientific Ltd, Stansted, UK). Ultrathin sections (70 to 90 nm thick) were obtained with a Leica EMUC7 ultramicrotome and transferred to 200-mesh copper TEM grids (SPI, West Chester, PA). Grids were stained with lead citrate and examined with an FEI Tecnai SPIRIT (FEI, Eidhoven, the Netherlands) TEM operated at 120 kV and equipped with a OneView camera (Gatan, Pleasanton, CA). Single-molecule fluorescence in situ hybridization was performed using hybridization chain reaction (HCR) version 3.0, as previously described32Choi H.M.T. Schwarzkopf M. Fornace M.E. Acharya A. Artavanis G. Stegmaier J. Cunha A. Pierce N.A. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.Development. 2018; 145: 1-10Crossref Scopus (299) Google Scholar with slight modifications. The probes for BiP and CHOP were both designed and ordered from Molecular Instruments (Los Angeles, CA). mRNA accession numbers are shown in Table 1. Briefly, tissue was fixed for 3 hours following sacrifice using 4% PFA/PBS freshly prepared with diethyl pyrocarbonate water. Then, solution was changed to 4% PFA/PBS/30% sucrose and was incubated by shaking overnight. The following day, the tissue was embedded in OCT and kept at −80°C until use. On the morning of the experiment, the tissue blocks were cut to produce sections (10 μm thick) using a cryostat, and kept in the cryostat chamber at −30°C until the beginning of the experiment. Then, tissue sections were warmed to room temperature and dried in a chemical hood for 7 minutes, followed by incubation in 70% ethanol/diethyl pyrocarbonate at 4°C for 1 hour. Then, sections were washed once in PBS and fixed in 4% RNase-free PFA for 7 minutes, washed with RNAse-free PBS, and permeabilized in 10 μg/mL proteinase K/PBS for 10 minutes at room temperature. Sections were then washed with RNAse-free PBS–Tween 0.1% twice, post-fixed with 4% RNase-free PFA for 5 minutes, and washed again with RNAse-free PBS–Tween 0.1% twice for 5 minutes. Then, sections were washed with acetylation buffer for 10 minutes, washed twice with RNAse-free PBS–Tween 0.1%, and rinsed in diethyl pyrocarbonate, as previously described by Shwartz and Zelzer.33Shwartz Y. Zelzer E. Nonradioactive in situ hybridization on skeletal tissue sections. Skeletal Development and Repair: Methods in Molecular Biology.in: Hilton M.J. Humana Press, Totowa, NJ2014: 203-215Google Scholar Next, sections were left to dry for 30 minutes at room temperature and equilibrated in HCR hybridization buffer (Molecular Instruments) for 10 minutes at 37°C. Probes were then added to the sections at a final concentration of 0.4 to 4 nmol/L and hybridized overnight in a humidified chamber at 37°C. The next day, the protocol described by Choi et al32Choi H.M.T. Schwarzkopf M. Fornace M.E. Acharya A. Artavanis G. Stegmaier J. Cunha A. Pierce N.A. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.Development. 2018; 145: 1-10Crossref Scopus (299) Google Scholar was applied using home-made wash buffer [50% formamide (75-12-7; Merck Millipore, Darmstadt, Germany), 5× saline-sodium citrate (Molecular Biology-P; 001985232300; Bio-Lab Ltd., Jerusalem, Israel), 9 mmol/L citric acid (pH 6.0; 77-92-9; Sigma Aldrich, Darmstadt, Germany), 0.1% Tween 20 (P1279; Sigma Aldrich), and 50 μg/mL heparin (H3393; Sigma Aldrich)]. The probe sets were amplified with HCR hairpins for 45 minutes at room temperature in HCR amplification buffer (Molecular Instruments). Fluorescently conjugated DNA hairpins used in the amplification were ordered from Molecular Instruments. Before use, the hairpins were snap cooled by heating at 95°C for 90 seconds and cooling to room temperature for 30 minutes in the dark. After amplification, the samples were washed in 5× saline-sodium citrate–Tween 20 and stained with DAPI (1:10,000; D9542; Millipore Sigma, Rehovot, Israel) diluted in PBS for 5 minutes and then mounted onto slides using Shandon Immu-mount.Table 1List of Probes Used for in Situ Hybridization Chain Reaction and Their Accession NumbersProbe nameAccession no.HCR amplifierAmplifier color, nmBiPNM_022310.3B5546CHOPNM_007837.4B3488Accession numbers from (last accessed June 7, 2022). Open table in a new tab Accession numbers from (last accessed June 7, 2022). Sections were imaged with a confocal LSM 800 microscope (Zeiss, Jena, Germany) at a resolution of 70 nm (x, y). The brightness of the in situ signal was enhanced in FIJI version 1.53c () for presentation in the figure.34Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T et al.Fiji: an open-source platform for biological-image analysis.Nature Methods. 2012; 9: 676-682Crossref PubMed Scopus (29116) Google Scholar For quantification of transcripts per cell, amplification was performed for 1 hour, and sections were imaged with the same microscope at a resolution of 70 and 400 nm (x, y, z). Autofluorescence imaging of cells was acquired with excitation at 488-nm laser, and cells were segmented with cellpose (green), using the cyto algorithm with a diameter size between 75 and 500 pixels, depending on the area in the enthesis. Images were then further quantified in CellProfiler version 3.1.9 () with custom pipelines.35Stirling DR Swain-Bowden MJ Lucas AM Carpenter AE Cimini BA Goodman A CellProfiler 4: improvements in speed, utility and usability.BMC Bioinformatics. 2021; 22: 433Crossref PubMed Scopus (54) Google Scholar Five sections of each zone (tendon end, mid-enthesis, and cartilage end) of three animals were measured; in total, there were 2228 cells of injured limbs and 2413 cells of control limbs. Achilles entheses were removed from 1-day postinjury mice using a surgical blade. At this age, the cartilaginous end marking the enthesis is distinguishable from the rest of the calcaneus by its bright white color. Measuring approximately 0.5 mm, the white extremity along with the attached tendon was isolated and homogenized. Total RNA was purified using the RNeasy Kit (Qiagen, Hilden, Germany). Reversed transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA), according to the manufacturer's protocol. Quantitative real-time PCR was performed using Fast SYBR Green master mix (Applied Biosystems) on the StepOnePlus machine (Applied Biosystems). Values were calculated using the StepOne software version 2.2, according to the relative standard curve method. For each gene, fold change relative to the control was calculated by delta-delta cycle threshold method. Data were normalized to 18S rRNA. Primer sequences are given in Table 2.Table 2List of Primers Used for the Quantitative Real-Time PCR and Their SequencesPrimer nameForward sequenceReverse sequenceBiP5′-GGGGACCACCTATTCCTGCGTC-3′5′-ATACGACGGCGTGATGCGGT-3′CHOP5′-TGTTGAAGATGAGCGGGTGGCA-3′5′-GGACCAGGTTCTGCTTTCAGGTGT-3′18S rRNA5′-GTAACCCGTTGAACCCCATT-3′5′-CCATCCAATCGGTAGTAGCG-3′ Open table in a new tab Gait and stride were assessed using the CatWalk XT 10.6 automated gait analysis system (Noldus Information Technology, Wageningen, the Netherlands) at 14, 28, and 56 days after manipulation (enthesis injury or sham operation). Mice were subjected to at least five runs in each assessment session. Following the identification and labeling of each footprint, gait data were generated. Functionality was assessed as in previous studies of recovery from nerve or enthesis damage28Moser H.L. Doe A.P. Meier K. Garnier S. Laudier D. Akiyama H. Zumstein M.A. Galatz L.M. Huang A.H. Genetic lineage tracing of targeted cell populations during enthesis healing.J Orthop Res. 2018; 36: 3275-3284Crossref PubMed Scopus (17) Google Scholar,36Deumens R. Jaken R.J.P. Marcus M.A.E. Joosten E.A.J. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection.J Neurosci Methods. 2007; 164: 120-130Crossref PubMed Scopus (137) Google Scholar, 37Kappos E.A. Sieber P.K. Engels P.E. Mariolo A.V. D'Arpa S. Schaefer D.J. Kalbermatten D.F. Validity and reliability of the CatWalk system as a
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