Tendinopathy, Tendon Healing & Tendon Regeneration
Alayna E. Loiselle, PhD Associate Professor IND 464 Musculoskeletal Basic Science Course October 21, 2019 Outline
• Review of homeostasis • Effects of Aging • Disruptions in homeostasis • Pathogenesis of tendinopathy/ tendinitis • Mechanisms of regeneration and healing !"#$%#&'()*%)+,-.-&,*"&#%.&,&/#('%*0&1%1/+,.(%#
23456(#&2788,9:&&234:;2788,9:&<=>? @"-.&A=2B@&C87D 0"1%" !"#$%(')*+',-./
!"#$%&$"'!"#$%(')*+',-./
;7%&5115';"<8'#4=#<1%&>5?'?"$"
23%4&$5%4'!"#$%(')*+',-./ +6"4&74'5$'"1(8'!%7*9%:(',-./ Tendon Cell Proliferation During Growth
Grinstein eLIFE 2019 Tendon Cell Proliferation During Growth
Grinstein eLIFE 2019 !"##$%&'%(%)*+
!"#$%&'()*+)(,%-,./0()%,1%%/(234452367 8*")$%9(&,+*-:;%'.#(2<= >%/9*/(&/9(+"-#$%().*:-.%-(?'*+(:&,.%/,-()*'/(@23 !%#-&0*(&-3%*-4 ,("-"*-".(&./& 0.10&-%#-"(2 Tendon Homeostasis Young Aged • Matrix composition and organization is not dramatically different • Decreased proteoglycan content • Changes in cell morphology Functional Consequences of Aging? Tendon is not mechanically sensitive to aging TT Fascicle TT FDL FCU Ackerman et al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ollagenase Induced Tendinopathy Induces degenerative changes via collagen degradation LD= low dose HD= high dose collagenase Orfei C et al. (2016) PLOS ONE 11(8): e0161590. Mechanically Induced Tendinopathy Low level Moderate High level • Fatigue loaded under anaesthesia Unloaded loading loading loading • Uphill treadmill running • Downhill treadmill running Induces matrix and cellular changes Fatigue Loading Induces Apoptosis N Andarawis-Puri et al., 2014. JOR Neviaser A. 2012. J Shoulder Elbow Surg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emodeling Balance Physiological exercise increases: proliferation collagen production tenocytic gene expression (w/o chondro/ osteo/adipo) Overuse/ Fatigue: Matrix Damage Tenocyte apoptosis Effects of pathology and/ or co-morbidities: promote degeneration +/- inflammation Smoking obesity high cholesterol Future Directions • Continue to identify co-morbidities that predispose or accelerate tendinopathy • Most clinical data are from late stage pathology • Beginning to use genetic animals models to better understand tendinopathy !"#$%#&'"()*#+ ! ,-../...&0"#$%#&1"2(*1&21%3"$41"5&2"1&6"(1 ! 78"1&9:.&;*))*%#&*#&(55%3*(0"$&<"()0<&3(1"&3%505 ! '"()*#+&*5&3%=2)*3(0"$&>6&53(1&?%1=(0*%# @%1=()&0"#$%# A3(1&!*554" B$<"5*%# !"#$%# C"$53(2"D3%= !"#$%&'#()*+,#-"&&./. ())(,#0"1"!2.34.5& ())6,#7"815&9% ())+ !"#$%&%'%()*(+*,-"./)%"/0*12(3-2'%-& Maximum Load at Failure !".%00-&*6-)7() 10 Control 8 40-5(2*6-)7() 6 [N] 4 2 0 14 21 28 35 42 49 63 Days Post-Surgery 94&"):%: &"):%: !"#$%&&%'%(')&*+',--.*'/01 2'3&#)$$"4 %(')&*+',--.*'/'566& 278$*' Similarities between wound and tendon healing Thiruvoth et al. 2015. Plastic and Aesthetic Research AJ Jomas et al. 2014. Advanced Drug Delivery Review Similarities between wound and tendon healing Nichols, Best, Loiselle. 2019 Inflammation Well-regulated inflammation is beneficial -activates healing cascade -recruitment/activation of cells Excessive/ Chronic inflammation is pathological -degenerative matrix changes -fibrotic healing Benefits of Anti-inflammatory therapy is controversial Timing may be key! Generally effective at preventing excess scar formation Early inhibition decreases mechanics Delayed healing maintains mechanics Cell-type specific considerations Virchenko et al., 2004. Am J Sports Med, Geary et al. 2015. PlosOne Proliferative/ Granulation Phase • Lasts a few weeks Col3a1 ISH • Begins ~day 7 in mouse model Scar • Proliferation of ‘fibroblasts’ Tendon Tissue • Bridging on injury site • Production of ECM components (Col1/ Col3) Tendon • Rapid deposition of disorganized ECM Day 3 Day 14 Thomopoulos et al., 2009. JOR, Thomopoulos et al., 2010. JBJS, Katzel et al. 2010. JOR, Awad Lab, Loiselle Lab Remodeling Phase • Lasts many months • Begins ~day 21 in mouse model • Reorientation of ECM • Mmp-mediated remodeling Day 21 Day 28 Day 35 "(##-11'()+F-2$.%L+M+=-).#$6-+9$)$/#-+3'+5-231.6.3/+$/5+%-;35-)./0 I-/53/+4/J(%K !"#$%&'())*+,-$)./0+%-123/1- 4;2$.%-5+0).5./0+'(/#6.3/+<+16%-/06, 4/#%-$1-5+6-/53/+1.7-8+9(): !"#$%&'&$( )"*$+",&(- =.13%0$/.7-5+>?6%$#-))()$%+@$6%.?+A>B@C >?#-11.D-+>B@+E%35(#6.3/ 4/1(''.#.-/6+>B@+F-;35-)./0 B3)G8+B3)H @$6%.?+@-6$))32%36-./$1-1 >B@+E%35(#./0+B-))1 @@E+E%35(#./0+B-))1 Double-edged sword of tendon healing Matrix deposition Mechanical properties Fibrosis • Thickening and/or scarring of connective tissue • Typically in response to injury • In response to injury fibrosis= scar tissue • Excess matrix deposition • Disorganized matrix Inflammation Matrix Deposition • Exuberant healing response !"#$%&'()'*+,&'+-./%,.'01,2+&3'$-.'43#,'!!'*+$1,&,2' (-'4,-.(-'5,$6+-7 @A#B$"3C$ !%($ ?&3* ()*'$ PQ5:*5N 9&5R*K'K DEFG!H !"#$%&'$()*'$+!%(,-$./0$12&3$45"6$4&'7$3*&8$2"8'5"3$ / .> LF ./ .@ >. >M 9):;$%&'$()*'$+9%(,-$(? J**BK (&NK$O"K'APQ5:*5N Body Weight 50 * LFD 40 HFD Body Weight Fasting Blood Glucose Glucose Tolerance Test Percent Body Fat 30 50 250 600 60 * LFD * * * **** 20 40 200 HFD * 400 40 10 30 150 Body Weight (g) Weight Body 0 20 mg/dL 100 200 LFD 20 LFD HFD Body Fat [%] 10 50 Diet (g) Weight Body HFD 0 0 [mg/dL] Glucose Blood 0 0 LFD HFD LFD HFD 0 15 30 60 120 LFD HFD Diet Diet Time Post-Glucose Bolus Diet S2B*56&8T O3"KU8*V$>/.FV T2DM Impairs Tendon Healing Gliding Resistance MTP Flexion Angle Max Load at Failure 80 ** 4 LFD 50 LFD HFD ** 40 HFD 60 3 30 40 * 2 20 20 1 10 0 0 Max Load at Failure [N] 0 10 14 21 28 [A.U.] Resistance Gliding 10 14 21 28 MTP Flexion Angle [Degrees] 10 14 21 28 Days Post- Repair Days Post- Repair Days Post- Repair Col3a1 Col1a1 6 4 LFD * * HFD * 3 4 * * 2 * mRNA Expression mRNA mRNA Expression mRNA 2 1 * Relative 0 Relative 0 3 7 10 14 21 28 3 7 10 14 21 28 + Ackerman PlosOne. 2017. Days Post-Repair Days Post-Repair !"#$%&'()(*+,%-*.%/)01',%2*3)-44-05(* F4/80 7 !"#$%&'()*&+ ,-./ Non-T2DM * 6 T2DM 012345555.678 012345555.678 * * 5 !"#$%# *!"#$%# 4 * 3 * 2 01234 Relative Expression Relative 1 * 0 7 14 21 28 Days Post-Repair 6+9):;'#< 7=">?#)@5-4AB@ &#'(")*"$+,)'(%-.)/"+'%#0"#0 1(%2%#/"$+,)'(%-.)/"+-("*"#'" &#'(")*"$3+-(%2%#/"$+-(%45#62),,)0%(7+89+,)'* &#'(")*"$3+")(27+)#0545#62),,)0%(7:+8;+,)'* Aging decreases fibrotic tendon healing (too much) Ackerman et al., JOR. 2017. Aging decreases fibrotic tendon healing (too much) No change in proliferative capacity Decreased matrix production on a per cell basis Is the cellular environment the same? Ackerman et al., JOR. 2017. 543 THE JOURNAL OF BONE & JOINT SURGERY · JBJS.ORG BIOLOGIC ASPECTS OF FLEXOR VOLUME 85-A · NUMBER 3 · MARCH 2003 TENDON LACERATION AND REPAIR ments in the treatment of these injuries. While investigations into the biology of tendon healing have shed some light on the mechanisms by which tendon tissue heals, there has been rela- tively little progress toward the biologic enhancement of the healing process after injury and repair. As a result, modulation of the process by which tendons heal remains an appealing, !"#$#%&'(&)*++,&-."#%$&/*%0'%&1*2+#%$3&4%5"#%,#6&7&895"#%,#6but not currently practicable, concept. 543 Biologic Solutions to Adhesion Formation: /:*&6*++.+2"&6';<'%*%5,&'(&5*%0'%&:*2+#%$&2"*&%'5&=*++&6:2"265*"#>*0 THE JOURNAL OF BONE & JOINT SURGERY · JBJS.ORG BIOLOGIC ASPECTS OF FLEXOR Historical Perspective 1,7,15-17 VOLUME 85-A · NUMBER 3 · MARCH 2003 TENDON LACERATION AND REPAIR It is well recognized that motion prevents scar formation . There is not a specific amount of motion that is needed, only 895"#%,#6&1*2+#%$ 4%5"#%,#6&1*2+#%$ a relative amount that is, enough to overcome the problems ments in the treatment of these injuries. While investigations of scarring and adhesion formation. If one could decrease the into the biology of tendon healing have shed some light on the amount of scarring associated with a given amount of mo- mechanisms by which tendon tissue heals, there has been rela- tion, less motion would be required. This approach would tively little progress toward the biologic enhancement of the eliminate the need for aggressive postoperative motion pro- healing process after injury and repair. As a result, modulation tocols that place the tendon repair site at risk for dehiscence. of the process by which tendons heal remains an appealing, If the ideal scar inhibitor could be found, the patient could but not currently practicable, concept. be treated with postoperative immobilization in a cast or splint until the tendon healed, allowing the repair to heal in a Biologic Solutions to Adhesion Formation: mechanically protected environment without concern about Historical Perspective !"#"$%&'(&)*+,--. adhesions. It is well recognized that motion prevents scar formation1,7,15-17. Historically, prevention of adhesion formation has been There is not a specific amount of motion that is needed, only attempted on two fronts. The first method has been to place a a relative amount that is, enough to overcome the problems physical and mechanical barrier between the healing tendon Fig. 4 of scarring and adhesion formation. If one could decrease the and the surrounding tissues. The rationale for this approach Schematic representation of extrinsic (A) and intrinsic (B) pathways of amount of scarring associated with a given amount of mo- is that limiting contact between the tendon and its sheath tendon ?'%*&;2""'=@&healing. (Adapted from: Gelberman RH, Vande Berg JS, Lund- M*,#0*%5&/*%'6D5*, B26"'<:2$*,@& tion, less motion would be required. This approach wouldA%0*(#%*0&B#+#*.diminishes the amount of adhesions around the repaired ten- borg GN, Akeson WH. Flexor tendon healing and restoration of the glid- eliminate the6#"6.+25#%$&6*++, need for aggressive postoperative motion pro- don that is, the tendon is allowed to heal to itself but not to 4%(+2;;25'"D&6*++, ing surface. An ultrastructural study in dogs. J Bone Joint Surg Am. tocols that place the tendon repair site at risk for dehiscence. CBD'(#E"'E+2,5,the sheath and surroundingF!CGBHI tissues. The various barrier ma- 1983;65:78-79.) 18 If the ideal scar inhibitor could be found, the patient could CG:*25:&terialsFJ"$KI& that have been tried include silicone , polyethylene be treated with postoperative immobilization in a cast or 19 20 21 C?2,*;*%5&;*;E"2%*membranes , alumina sheaths , polytetrafluoroethylene , and splint responseuntil the observedtendon healed, clinically allowing is a combination the repair to of heal extrinsic in a and chondroitin sulfate-coated polyhydroxyethyl methacrylate mem- F+2;#%#%I mechanicallyintrinsic protected mechanisms environment (Fig. 4)1. without concern about branes22, among many others. In spite of the many different adhesions. The differences between the extrinsic and intrinsic mech-CL materials studied for this purpose, none are in routine clinical G69C+#%*2$* anismsHistorically, extend prevention beyond the of site adhesion of origin formation of the healing has been cells. The use at this time. attemptedextrinsic on two mechanism fronts. The appears first methodto be active has beenearlier to in place the healinga In a similar approach, many authors have attempted to physical and mechanical barrier between the healing tendon 10,11 Fig. 4 sequence, whereas the intrinsic mechanism is often delayed . use chemical modulation to diminish the amount of scar for- and the surrounding tissues. The rationale for this approach Schematic representation of extrinsic (A) and intrinsic (B) pathways of Some studies have demonstrated that the synovial sheath reacts mation after repair. The chemical agents that have been used is that limiting contact between the tendon and its sheath 23,24 25,26 tendon healing. (Adapted from: Gelberman RH, Vande Berg JS, Lund- with a greater proliferative and inflammatory response com- in these efforts include local and parenteral corticoste- diminishespared thewith amount the endotenon of adhesions and aroundtendon properthe repaired12. Other ten- studies roids, dimethyl sulfoxide27, beta-aminoproprionitrile28, hyalu- borg GN, Akeson WH. Flexor tendon healing and restoration of the glid- don havethat shownis, the tendonthe synovial is allowed fibroblasts to heal to to be itself more but reactive not to to cy- ronic acid29,30, and 5-fluorouracil31,32, among many others. The ing surface. An ultrastructural study in dogs. J Bone Joint Surg Am. the sheathtokines and and surrounding to have a greatertissues. capacity The various for degradation barrier ma- of the common principle of these methodologies is reduction of in- 1983;65:78-79.) 18 terialsextracellular that have beenmatrix tried11. In includeaddition, silicone it is thought, polyethylene that predomi- flammation. In the case of the corticosteroids and hyaluronic membranesnance19 of, alumina the extrinsic sheaths mechanism20, polytetrafluoroethylene of healing leads 21to, andincreased acid, the goal is to diminish inflammation by inhibiting lym- response observed clinically is a combination of extrinsic and chondroitincollagen sulfate-coated content at thepolyhydroxyethyl injury site as methacrylatewell as to a reduction mem- in phocyte migration, proliferation, and chemotaxis as well as intrinsic mechanisms (Fig. 4)1. branesthe22, amonglevel of collagenmany others. organization In spite and of materialthe many properties different of the macrophage motility. Similarly, 5-fluorouracil, an antimetab- The differences between the extrinsic and intrinsic mech- materialsreparative studied tendon for this tissue purpose,13,14. For none these are reasons, in routine it is clinicalbelieved that olite, suppresses scar formation by inhibiting contraction of anisms extend beyond the site of origin of the healing cells. The use at thethis predominance time. of the extrinsic mechanism of healing leads collagen lattice and proliferation of inflammatory cells31,32. extrinsic mechanism appears to be active earlier in the healing toIn scara similar formation approach, and adhesionmany authorss between have the attempted tendon andto sur- Researchers using barrier and chemical techniques have sequence, whereas the intrinsic mechanism is often delayed10,11. use chemicalrounding modulation peritendinous to diminish structures. the amount of scar for- reported some degree of adhesion reduction in laboratory and Some studies have demonstrated that the synovial sheath reacts mation afterModulating repair. The the chemical healing agents process that to haveenhance been the used intrinsic clinical studies of tendon repair18,20,21,23,25,29,31,32. In spite of these with a greater proliferative and inflammatory response com- in thesepathway efforts (andinclude augment local23,24 end-to-end and parenteral tendon25,26 corticoste- healing) while findings, these methods are not widely used in clinical prac- pared with the endotenon and tendon proper12. Other studies roids, suppressingdimethyl sulfoxide the extrinsic27, beta-aminoproprionitrile pathway (and diminishing28, hyalu- adhesions tice. This suggests that none of them have been demonstrated have shown the synovial fibroblasts to be more reactive to cy- ronic ofacid the29,30 tendon, and 5-fluorouracil to surrounding31,32, tissues)among manycould others.lead to Theimprove- to be effective in most clinical settings. tokines and to have a greater capacity for degradation of the common principle of these methodologies is reduction of in- extracellular matrix11. In addition, it is thought that predomi- flammation. In the case of the corticosteroids and hyaluronic nance of the extrinsic mechanism of healing leads to increased acid, the goal is to diminish inflammation byDownloaded inhibiting lym-from www.ejbjs.org on October 7, 2006 collagen content at the injury site as well as to a reduction in phocyte migration, proliferation, and chemotaxis as well as the level of collagen organization and material properties of the macrophage motility. Similarly, 5-fluorouracil, an antimetab- reparative tendon tissue13,14. For these reasons, it is believed that olite, suppresses scar formation by inhibiting contraction of the predominance of the extrinsic mechanism of healing leads collagen lattice and proliferation of inflammatory cells31,32. to scar formation and adhesions between the tendon and sur- Researchers using barrier and chemical techniques have rounding peritendinous structures. reported some degree of adhesion reduction in laboratory and Modulating the healing process to enhance the intrinsic clinical studies of tendon repair18,20,21,23,25,29,31,32. In spite of these pathway (and augment end-to-end tendon healing) while findings, these methods are not widely used in clinical prac- suppressing the extrinsic pathway (and diminishing adhesions tice. This suggests that none of them have been demonstrated of the tendon to surrounding tissues) could lead to improve- to be effective in most clinical settings. Downloaded from www.ejbjs.org on October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ellular Basis of Healing: Extrinsic Cells Bone Marrow Ablation Bone Marrow Harvest GFP C57BL/6 Bone Marrow Transplant GFP Bone Marrow Chimeric Mouse Tendon Injury and Repair Fluorescence !"##$#%&'(%)*)'+,'-"%#*./0'123&*.)*4'!"##) (+."'9%&&+:'5"&*;"<'!"##)'9*/&%3"'=>"4*,*4%##6'3+'3?"'@">%*&'=*3" !+.3&+# 5%6'78 ! =>"4*,*4'=$EB>+>$#%3*+.)'&"F%*.'$.G.+:. ! H$.43*+.'.+3'I4#"%& !"#$%# !"#$%# !"#$%# &'()*!+,,-" ! DEFEGHI*,->>+'+"#0*>%)*($@",+%#*>%)G(0+%# A.B*.C$&"< 5%6'D .%+,"//"*"0*(/12*34531*6/%,7#"2*8(9+:(;(2*<1*"0*(/1*=%-)#(/*%>*?"//*6@A,+%/%BA1*344C Future Work • Delineate intrinsic vs. extrinsic contributions of cell types – Macrophages – S100a4 • Define functions of intrinsic & extrinsic populations • Understand how homeostatic populations change in response to injury !"#"$"%&'()"*+"&,($#-*./0%12$(3 4"'&,*56""7 8&'9%"*56""7 ($<9%"= ($<9%"= :$; :$; >$<9%"= >$<9%"= !"#"$%&'(&)* +,-".-)(/0-1223/-4**)(5-67-!&68"$&9)(-:*;&*""#&*; !"#"$"%&'()"*+"&,($#-*./0%12$(3 789#:;*&<-)*$-)$=(.-."*$;*-&8>()*."$-&*-.;-.?"- 9)<'-.;-$"."#8&*"-&@-"89#:;*&<-."*$;*A-<;=($- ?")(-A<)#B("AA(: &*-)*-)$=(.-"*C&#;*8"*. D$=(. 789#:;*&< 789#:;*&< E-F""' E-F""' G-F""'A !"#"$%&'(&)* +,-".-)(/0-1223/-456 !"#"$"%&'()"*+"&,($#-*."/$&'&, !"#$%%&$'&(%)*&+,-.&/012$3 !"#"$"%&'()"*+"&,($#-*."/$&'&, 0%&1($#*2,(34,($"&#"*1",,5*&6'"%*"$'7"5(5 ($89%: :,69%'%- 2,(; ;6<9=)%><-' !"#$%&'()*+),')%-./)0123/)4,5,-678,9' Where do we go from here? (2017) üCritical mass of labs studying cell lineage/fate/function üBig data and single-cell RNAseq • Development of tendon specific tools • Identification of new ‘tendon-specific’ markers >="+"&$%&?"&,%&#%?@ !"#$%#&'"((&)"*"+%,"#"-*. ! !"#$%#&'"((&)"*"+%,"#"-*. ! /"(-#"0*"&12#3*-%#4&%1&4256%62(0*-%#4 ! 7""$&*%&$"1-#"&3"((&%+-,-#8&(-#"0,"&0#$&10*" ! 9#$"+4*0#$-#,&%1&3"((:3"((8&3"((:;0*+-<&-#*"+03*-%#&!6="#%*.6" 9#$"+4*0#$-#,&'"((:'"((&A#*"+03*-%#4 /"1-#-#,&'"((&D-#"0,"&0#$&!"+;-#0(&C0*" A;;2#"&'"((4 A#E2+. B"4-$"#*&!"#%3.*"4 FGHH0I(-#"0,"JK FGHH0I03*-L":K&MFNOJ N.%1-5+%5(04*4 FGHH0I(-#"0,"JK FGHH0I03*-L"J FGHH0I(-#"0,"JK FGHH0I03*-L"JK&MFNO: 7%#:;.%1-5+%5(04*4 C-5+%*-3&)"0(-#, Tenocyte transition to myofibroblasts? Ø ”Specialized”/ Activated Fibroblasts Ø Involved in matrix deposition Ø Restoration of tissue integrity !"#$%#&'#()*+ ;4)2"&'#8913312-%# <=4"//&<>?& ,-.*%/-/ @":%/-2-%# 0%&!*"123"#2/ !"#%4+2"&5"2"*%6"#"-2+ 7$-88"*"#2-19&*"/:%#/"&2%&-#()*+ >5*%#-4&9%B79"C"9&-#8913312-%# ?14*%:516"&A"*/-/2"#4" ,-.*%/-/ ?+%8-.*%.91/2&A"*/-/2"#4" <=4"//&<>?&A*%$)42-%# Fibroblast heterogeneity at baseline with differential contributions to myofibroblast fate Am J Respir Cell Mol Biol, 2019 https://www.atsjournals.org/doi/abs/10.1165/rcmb.2018-0313OC !"#$%&'()*+,$-+./$*&)%$,0+1").&1+%2#3+&)4# !"#$%&'#$()*+ '=05(*#7>?$"5@$A-5?*>(5"B$)=>A*)$(C0.$*>D0 !"#$%&$'()*+,-./0.# !"#$12$'()*+,-./0.# 9+,34$$$ '()*5 !4': 9+,34$$$ '()*5 !4': '()*5 ,34$ ! 4 50 !m < 50 !m 9+,34$$$6)7&8 !4': 9+,34$$$6)7&8 !4': ,34$6)7&8 ! ; 50 !m ! 50 !m !"#$%&'()&*#)+,-.$)#.-(,&'+",/$0#(1##'$2344-5$-'6$ 7289 J#=,','D$("#$<;&=,0)&0.-+($/)#7%C)+&)+$6C),'D$(#'6&'$"#-.,'D ! HI8$J#/&+,(,&' HI8$I&'()-%(,&' EF,0)&0.-+(?$(#'&%;(#G 8;&=,0)&0.-+(+ 2344-5 @7289 <#)D# ! :#-).;$-..$6#)<-.$=,0)&0.-+(+$-)#$2344-5>?@7289> ! :&$%&7.&%-.,A-(,&'$+##'$,'$BC,#+%#'($&)$-%(,*-(#6$ "#/-(,%$+(#..-(#$%#..+ !"#$%%$&'($% $#)*+,)-./0)12345236515,)782119 S100a4+ cells are not myofibroblasts in the healing tendon But, S100a4-lineage cells become α-SMA+ myofibroblasts 80 ** 60 40 20 Fluorescent area [%] 0 S100a4Lin+; S100a4-GFPpromoter+; α-SMA+ α-SMA+ Ackerman et al., eLife. 2019 (1C%03"'J.C&A34F&3.C'.2'!"#B3C %CD'!"# K#-A@//3.C'&.'=1.234A.4?%/&'6%&@ 5@-%3A H%AI@/& !"?@A%#3/93:)*+), =3"@ & !" B%4@?/'[email protected]'"@??/'&E%&'@#-A@//'!"# )*+),' #$% D%1/'-./&+/FAG@A1 !"#$ %&'()*+),'-./&+.-' 01.234/ ! 567'8!"#93:;