Natural Large‐Scale Regeneration of Rib Cartilage in aMouse ModelMarissa K Srour,1 Jennifer L Fogel,1 Kent T Yamaguchi,1 Aaron P Montgomery,1 Audrey K Izuhara,1

Aaron L Misakian,1 Stephanie Lam,1 Daniel L Lakeland,1 Mark M Urata,2 Janice S Lee,3�

and Francesca V Mariani1

1Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California,Los Angeles, CA, USA

2Division of Plastic and Reconstructive Surgery, Children’s Hospital Los Angeles, Los Angeles, CA, USA3Department of Oral and Maxillofacial Surgery, University of California, San Francisco, San Francisco, CA, USA

ABSTRACTThe clinical need for methods to repair and regenerate large cartilage and bone lesions persists. One way tomake new headway is tostudy skeletal regeneration when it occurs naturally. Cartilage repair is typically slow and incomplete. However, an exception to thisobservation can be found in the costal cartilages, where complete repair has been reported in humans but the cellular andmolecularmechanisms have not yet been characterized. In this study, we establish a novel animalmodel for cartilage repair using themouse ribcostal cartilage. We then use this model to test the hypothesis that the perichondrium, the dense connective tissue that surroundsthe cartilage, is a tissue essential for repair. Our results show that full replacement of the resected cartilage occurs quickly (within 1 to2 months) and properly differentiates but that repair occurs only in the presence of the perichondrium. We then provide evidencethat the rib perichondrium contains a special niche that houses chondrogenic progenitors that possess qualities particularly suitedfor mediating repair. Label‐retaining cells can be found within the perichondrium that can give rise to new chondrocytes.Furthermore, the perichondrium proliferates and thickens during the healing period and when ectopically placed can generate newcartilage. In conclusion, we have successfully established amodel for hyaline cartilage repair in themouse rib, which should be usefulfor gaining amore detailed understanding of cartilage regeneration and ultimately for developingmethods to improve cartilage andbone repair in other parts of the skeleton. © 2014 American Society for Bone and Mineral Research.

KEY WORDS: CARTILAGE REPAIR; PERICHONDRIUM; SEGMENTAL DEFECT; CHONDROGENIC PROGENITORS; CHONDROCTYES; STEM CELLS

Introduction

Although humans are able to repair small skeletal injuriesfairly well, the ability to repair large defects is limited. Each

year, millions of patients undergo bone‐ and cartilage‐replace-ment surgeries to restore structure and function because ofinjuries, skeletal birth defects, and cancerous lesions.(1) Recon-struction may involve bone or cartilage autografts, morsellizedbone, implanted scaffolds, and for the largest of defects,composite tissue‐free flaps with implanted materials such astitanium rods, plates, and screws. The use of allografts poseschallenges because of immunological rejection, infection at thesite, resorption, and/or shortages of material. Autogenousgrafting is possible, but the amount of material is limited, asecond surgical site is needed, and the donor site can be subjectto long‐termmorbidity factors.(2) Prosthetic devicesmay not lastthe patient’s life span and can cause significant inflammation.Thus, although there is much success to celebrate with these

approaches, significant challenges still exist and new methodsto stimulate skeletal repair are critically needed.

Since the early part of the 20th century, the ability of thehuman rib to regenerate itself has been appreciated.(3–5)

However, scientific reports demonstrating repair have beensporadic and anecdotal. Currently, this phenomenon is besttaken advantage of by craniomaxillofacial surgeons, who useboth cartilage and bone material from the rib for jaw, face, andear reconstruction.(6,7) Understanding how this repair occurscould be instrumental for developing strategies to repaircartilage and bone in other locations in the body, and morecomprehensive studies of rib repair in humans are needed. Todissect the potentially unique cellular and molecular mecha-nisms involved, however, the development of an animalmodel isa key first step.

All types of cartilage (hyaline, elastic, and fibrocartilage) canresist crushing or stretching but have limited repair capacitypossibly because of being avascular and/or consisting largely

Received in original form February 4, 2014; revised form July 28, 2014; accepted July 30, 2014. Accepted manuscript online August 20, 2014.Address correspondence to: Francesca Mariani, PhD, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine,University of Southern California, 1425 San Pablo Street, BCC 407, Los Angeles, CA 90033, USA. E‐mail: fmariani@usc.edu�Present address: NIDCR/NIH, Bethesda, MD, USAAdditional Supporting Information may be found in the online version of this article.

ORIGINAL ARTICLE JJBMR

Journal of Bone and Mineral Research, Vol. 30, No. 2, February 2015, pp 297–308DOI: 10.1002/jbmr.2326© 2014 American Society for Bone and Mineral Research

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of terminally differentiated cells. However, there are someindications that stem cell niches might exist within theintervertebral disc(8) and the knee joint,(9) which may be utilizedto facilitate repair or homeostasis. Little is known regarding thelocation of possible stem cell niches in costal cartilages.

The perichondrium and periosteum are fibrous sheaths ofvascular connective tissue surrounding the rib cartilage andbone segments, respectively. Reports in humans have indicatedthat both the costal cartilage and bonewill regenerate over timewhen this connective tissue is left intact.(2,6,7,10,11) The role of thesurrounding connective tissue during cartilage repair may bemultifaceted—providing vascular supply, producing necessarychondrogenic‐inducing factors, and/or providing a niche orscaffold for repair. However, a central component for cartilagerepair may be the ability of stem cells residing within the tissueto produce chondrogenic progenitors.

Indeed, a number of studies have indicated that this sheathharbors the requisite progenitor stem cells used for repair (forbone(12,13) and cartilage(14–16)). Here, to better understand theability of the rib to regenerate, we first revisit the ability ofhumans to regenerate costal cartilage using high‐resolutionimaging. We then take advantage of the similarity betweenmouse and human thoracic anatomy (Fig. 2) to develop amousemodel for rib regeneration, where we can specifically test therole and requirement of the perichondrium in costal cartilagerepair.

Materials and Methods

Case study

UCSF’s Committee on Human Research–approved consent (CHR#H42089‐22594) was obtained, allowing for postoperativequantitative CTs to examine the regeneration at the graftharvest site. CT imaging was performed with a 16‐row multisliceCT scanner (Lightspeed, GE, Milwaukee, WI, USA). The kVp andmA were based on patient weight. A 3.75‐mm image slicethickness was obtained and reconstructed to a 1.25‐mmthickness. The CT image field of view was restricted to theresection site marked by a radio‐opaque skin marker to limitradiation exposure. A bone density phantom (MINDWAYS,Austin, TX, USA) was included. The DICOM data set was analyzedand reconstructedwith Amira (VSG/FEI, Burlington,MA, USA) andInVivo (Anatomage, San Jose, CA, USA) software. Our studycomplies with the World Medical Association Declaration ofHelsinki, Ethical Principles for Medical Research Involving HumanSubjects.

Mouse procedures

CD‐1, FVB (Charles River Laboratories, Wilmington, MA, USA), orMRL/MpJ (000486, Jackson Laboratory, Bar Harbor, ME, USA)mice (average of 53 days of age) were used for a survival surgeryprocedure in which a costal cartilage segment was excised withor without the perichondrium. The length of the resectedcartilage varied from �2 to 5mm with an average of 4.10mm(representing about 33% of the typical length of costal cartilagesfrom ribs 8 to 10). The procedure took �1 hour, could be easilycarried out after practice, used commercially available microsur-gical tools, and was well tolerated. For autograft surgeries, a flapincisionwasmade in the intercostalmuscles between rib 7 and 8,and a 4‐ to 5‐mm perichondrium strip from ribs 8, 9, or 10 wasinserted either as an autograft or using tdTomato labeled

(C57BL/6J, 007676, Jackson Laboratory) tissue into a C57BL/6Jmouse.

Further details for surgical procedures can be found in theSupplemental Materials. To assess new calcium deposition, micewere given an intraperitoneal injection of calcein (Sigma C0875,Sigma, St. Louis, MO, USA) 12.5mg/kg, 2 days beforeeuthanization, and incorporation was assessed using a ZeissAxio Observer fluorescent microscope (Carl Zeiss Microscopy,Thornwood, NY, USA). For label retention studies, 10‐day‐oldfemale CD‐1 mice were injected intraperitoneally with 50mg/kgof 5‐ethynyl‐2’‐deoxyuridine (EdU), twice daily for 3 days. Thispulse was followed by a chase of 8 hours, 2 weeks, or 8 months.EdU was then visualized using Click‐iT (Life Technologies,Carlsbad, CA, USA), and labeled cells were distinguished fromautofluorescing red blood cells because they were positive for anuclear stain (Hoechst 33258, Life Technologies). All procedureswere carried out in accordance with approved protocols by theInstitutional Animal Care and Use Committee (IACUC) at theUniversity of Southern California (USC).

Tissue analysis and histology

Rib cages were fixed in 95% EtOH and stained with alizarin redand alcian blue using a standard protocol(17) or fixed in 4%paraformaldehyde and decalcified (3 to 10 days in 10% EDTA) forhistological analysis. Samples were prepped for paraffin or cryo‐sectioning using standard protocols or for plastic sectioningusing the Immuno‐Bed Kit (Polysciences, Inc., Warrington, PA,USA; #17324). Sections were stained with nuclear fast red,hematoxylin and eosin, and alcian blue, using standardprotocols or trichrome (Gomori One Step, aniline blue,Newcomer Supply, Middleton, WI, USA; 9176B). After antigenretrieval, sections were incubated in antibody for proliferatingcell nuclear antigen (PCNA) (Vector Labs, Burlingame, CA, USA;VP‐P980, 1:100) followed by detection with Alexa Fluor 568 goatanti‐mouse IgG (Hþ L) secondary (A11031, 1:300). Images werecaptured with a Nikon AZ100 macroscope (Nikon Corporation,Tokyo, Japan) or a Zeiss Axio Imager (Carl Zeiss Microscopy).

Micro–computed tomography (mCT)

Scans of the mouse rib cage were performed at the USC’sMolecular Imaging Center on a subset of the animals (80 to293 days after resection). mCT scans were acquired with theInveonCT scanner (Siemens Medical Solutions USA, Inc., Knox-ville, TN, USA) with the following settings: 80 kVp, 120 uA, nofilter, 451 projections covering 220 degrees with 4s/projection,bin¼ 2, and a voxel size of 18.729 microns. Data werereconstructed using Cobra Reconstruction Software (ExximComputing Corp., Pleasanton, CA, USA). One sample wasscanned with higher resolution on the mCT 50 scanner (ScancoMedical AG, Bruttisellen, Switzerland) using the followingsettings: 70 kVp, 114 uA, no filter, 2000 projections covering360 degrees with 1.5s/projection, and a voxel size of 6.8 microns.Higher‐resolution data were reconstructed using the Scancosoftware. Data sets were loaded into Amira 5.3.1 (Visage Imaging,Inc., Berlin, Germany) or Osirix (http://www.osirix‐viewer.com) forvisualization and analysis. Images were segmented to measuremineralized cartilage volumes (mm3) in areas of resection.Measurements of each sample were performed by at least twopeople blinded to the strain and healing time.

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Quantification and analysis

To assess healing over time and across mouse strain, weconstructed a mathematical model that considered physical andgeometric parameters. To determine the resection volume andsurface area should repair be complete and continuous, we usedthe resection length and the major and minor radii of the cutends (clearly identifiable), as measured from reconstructed mCTscans, and assumed a tapered elliptical cylinder profile. We thenused the measured repair volume and assumed a simpledifferential equation, dC/dt¼ (Vf‐C)/(sb[Vr/Sr]), where C is repairvolume, t is time, Vf is final repair volume, Vr is the resectionvolume, Sr is the resection surface area, and sb is a parameterdefined in units of days/mm to express the inverse rate or“slowness” of repair for each genetic background, b. The modelwas fit using the Bayesian modeling language JAGS via statisticalsoftware, R. Further details are explained in SupplementalMaterials.

Results

A case study in a human male

Although regeneration of the costal cartilage in humans hasbeen described, the patients in these studies were either unusual(undergoing reconstructive surgery for pectus excavatum) or

the radiographic imaging used was at low resolution.(6,7,11) We,therefore, decided to determine if regeneration could bedetected firsthand in our clinic. A 42‐year‐old male requiredcraniofacial reconstruction at UCSF’s Department of Oral &Maxillofacial Surgery because of trauma. We harvested a portionof bone (8 cm) and cartilage (1 cm) from the right 6th rib (Fig. 1A),and a CT scan immediately after surgery was used to estimate theresection site (Fig. 1B, B0). After �6 months, a reconstructed CTscan from the same patient showed incomplete regeneration ofthe bone and regeneration of the costal cartilage with a distinctlow‐density region between the two segments likely represent-ing the reformation of the costochondral joint (Fig. 1C, C0).Although this imaging provides evidence for both bone andcartilage repair in a human and confirms previous and anecdotalreports, understanding the cellular and molecular mechanismnecessitates histological and cellular analysis. This is bestachieved by establishing an animal model.

Development of a mouse model

We devised a survival surgery procedure in mice that takesadvantage of the observation that mouse and human thoracicanatomy are very similar (Fig. 2A–E) and that allowed for theremoval of cartilage portions with and without the surroundingperichondrium (Fig. 3A–F0).

Fig. 1. Case study: costal cartilage repair in a 42‐year‐old male. (A)Schematic, anterior view of the human thoracic cage. The region to beresected is indicated by a rectangular box. (B, B0) A 3D reconstructed CTimage was taken immediately postoperatively, time 0. Red and blue linesindicate the approximate lengths of bone (8 cm) and cartilage (1 cm),respectively, that were resected. A radiopaque marker (white dot) wasplaced on the skin incision to identify the resection site and limit FOV andradiation exposure. (C, C0) A 3D reconstructed CT image taken atapproximately 6 months postoperatively showing incomplete bone repair,cartilage repair, and restoration of the costochondral joint (yellowarrowhead).

Fig. 2. Mouse thoracic anatomy. The rib cage consists of a skeletalframework that protects the heart and lungs and is composed of threeparts: the thoracic vertebrae, ribs, and sternum. (A) A 3/4‐view diagramshowing that in mice, as in other land animals, the rib has two segments,a proximal bony portion (depicted in red) and a distal cartilage portion(blue). Ribs 1 to 7 (true ribs) articulate with the sternum, whereas thefalse ribs, 8 to 10 (arrows) and 11 to 13 do not. Humans have a similarorganization except that ribs 8 to 10 are fused together distally and twopairs of floating ribs are present instead of three. (B) A hematoxylin andeosin–stained cross section showing that as in humans, the bony portionhas a central bone marrow cavity and is surrounded by a periosteum. (C)Hematoxylin and eosin–stained cross section through costal cartilage.(D) A near adjacent serial section of the same rib, alcian blue. (E) Anotheradjacent serial section stained with Gomori’s trichrome. The costalcartilage has typical hyaline features, including isogenous groups and arich proteoglycan matrix. The surrounding perichondrium is easilydistinguished because of its acidophilic (C) and collagen‐rich matrix (E)and because it contains cells with spindle‐shaped nuclei. Lines in Aindicate locations of sections in B and C–E. Minor adjustments weremadewith the Photoshop Levels tool to correct for underexposure. (B–E)Scale bar¼ 100 microns.

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Fig. 3. Costal cartilage excision procedure. (A) Diagram depicting costal cartilage removal and the retention of the perichondrium in the animal(cartilage‐only surgery). An incision through the skin (brown), fat (yellow), and muscle layers (red) is followed by an incision in the perichondrium (gray)along the length of the resected region and then the cartilage (blue) is excised. The red blocks represent the intercostal muscles. Care is taken to preventtearing the thoracic lining (green), which rostral to the diaphragm will create a pneumothorax. (B) After the perichondrium is incised on the superficialsurface, the excised cartilage (white arrow) is cut at one end (dashed line), lifted away from the perichondrium (yellow arrow), and cut at the other end(dashed line). (C) Hematoxylin and eosin–stained cross section of a resected specimen fixed immediately after closing. Note that the perichondrium(outlinedwith a white dotted line) is present and, apart from the incision to remove the cartilage, is intact. A neighboring rib is shown for comparison. (D)Removed cartilage with smooth exterior. (D0) Plastic section (neutral fast red) of excised cartilage (blue bracket) showing absence of the perichondrium.(E) A diagram illustrating removal of both the cartilage and perichondrium. (F) Removed cartilage with rough exterior (yellow arrows). (F0) Plastic section(neutral fast red) of removed cartilage (blue bracket) showing the presence of the perichondrium (yellow bracket). (G) Skeletal preparations (CD‐1) of thethoracic region stained with alizarin red and alcian blue at E18.5. (G0) Skeletal preparation at �2 months of age (time of surgery) showing that in mice,costal cartilages are completelymineralized by this time. (G00) Skeletal preparation at�11months of age (late analysis time point). Repair of an incised ribcould be detected when the perichondrium is retained in the animal (blue arrowheads, an enlarged image of this repair is found in Fig. 4B). (H, I)Mineralization was visible with alizarin red/alcian blue staining and with high‐resolution mCT scanning (unresected costal cartilage). (B, D, F) Scalebar¼ 1mm; (C, D0, F ’, H, I) scale bar¼ 100 microns. All micrographs: minor adjustment with Photoshop Levels to correct for underexposure.

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Because we did not know if wild‐type mouse strains wouldundergo cartilage repair at all, we decided also to include MRL/MpJ mice in our study. This line has an enhanced regenerativecapacity in some tissues.With regard to cartilage, a punchwoundin the ear elastic cartilage of MRL/MpJ mice led to fullregeneration after 4 to 5 weeks, whereas a punch wound inthe control strain remained intact.(18) In addition, MRL/MpJ miceappear to have a better capacity to repair articular injuries(19) anda lower immune response after a traumatic articular injury,suggesting that they may be protected from posttraumaticarthritis.(20) To date, the capacity for costal cartilage repair inMRL/MpJ mice has not been investigated.To test the role of the perichondrium, surgeries were divided

into three groups: 1) surgeries in which only the costal cartilagewas removed, leaving the perichondrium remaining in theanimal (n¼ 33, Fig. 3A); 2) surgeries in which both rib andperichondrium were removed (n¼ 12, Fig. 3E); and 3) controlseuthanized shortly after surgery to assess technique (n¼ 7).Aside from the incision made to remove the cartilage, theremaining perichondrium was intact and relatively undisturbed(still attached to surrounding tissues) (Fig. 3C). The vast majorityof the cartilage had been removed, although a few smallfragments intimately attached to the perichondrium wereoccasionally left behind. The integrity of removed cartilageportion was also analyzed. It typically had a smooth exteriorunder surface illumination, and histological analysis revealed noattached perichondrium (Fig. 3D, D0). Upon visual inspection ofwith‐perichondrium portions, the cartilage surface had a distinctragged appearance, and an intact perichondrium was clearlyevident in histological section (arrows and bracket, Fig. 3F, F0).Our human case study along with a few published reportsindicated that healing might take on the order of severalmonths.(5,6) Thus, within the cartilage‐only surgeries,we examined approximately half at 1 to 3 months (average95 days, n¼ 24). The other half were long‐term recoverysurgeries analyzed at 7 to 9 months (average 274 days,n¼ 13). The complete‐removal surgeries had an averagepostsurgical recovery time of 117.5 days, n¼ 19. Roughly halfof the surgeries utilized the MRL/MpJ mouse strain (n¼ 26/56).At birth, the costal cartilages of mice can be detected with

alcian blue (Fig. 3G). However, by sexual maturity, when thesurgeries were performed (average age¼ 53 days), chondro-cytes become entrapped in a mineralized matrix (Fig. 3G0). Thismineralization was retained in older mice (9 months, Fig. 3G00)and at high magnification was evident as a spongiform matrixboth by alizarin red staining and high‐resolution mCT (Fig. 3H, I)

with the empty spaces likely occupied by resident hypertrophicchondrocytes. This mineralization greatly facilitated our abilityto locate the resected region and additionally to quantify repairby mCT scanning (Fig. 3G00, arrowheads). This phenomenon is incontrast to humans whose costal cartilages only mineralize laterin life. Indeed, the degree of mineralization can be used todetermine the age of forensic specimens.(21)

Surgeries in which both cartilage and the surroundingperichondrium were excised failed to repair even after 9 monthsas assessed by whole‐mount skeletal staining and mCT (Fig. 4A,A0). Occasionally scattered mineralized fragments were evident,but these were small and/or disorganized (data not shown).To our surprise, however, all cartilage‐only surgery samples,regardless of genetic strain, repaired—with the majority of newcartilage filling in by 1 to 2 months followed by mineralization(Fig. 4B, B0). After 1 week post‐resection (PR), some alcian blue–positive cartilage could be detected at the lateral edges of the cutends (Fig. 4C). By 3 weeks PR, diffuse alcian blue staining wasevident along the resected region (Fig. 4D), whereas with longerhealing times, alizarin red staining was also present but typicallyfound in distinct patches of varying sizes (Fig. 4E; Fig. 5A, B).Samples with longer healing time points correlated with alizarinred staining that spanned a larger portion of the resection (Fig. 5).To definitively confirm that the repair tissue consisted ofcartilage,we serially sectioned through a healed region identifiedby skeletal preparation to examine the cellular profile (Fig. 4E).We found that the repair tissue did indeed consist of cartilage,extended throughout the resected region, and containedchondrocytes arranged in clusters typical of isogenous groups(Fig. 4G1–3). When comparing the new cartilage to unresectedcartilage (Fig. 4F), we noticed that new cartilage containedabundant hypertrophic profiles that at times appeared largerthan normal and that the surrounding perichondrium wasirregular and on average 2.10 times thicker (Fig. 4G1–3). Toensure that the observedmorphologywas not an artifact, we alsoanalyzed specimens prepared using standard histologicaltechniques (example in Fig. 4H–I0). We confirmed our initialfindings and additionally observed that, although thickened, theperichondrium histomorphology was normal—cells in theperichondrium were surrounded by matrix, had somewhatelongated nuclei, and were not hypertrophic (Fig. 4H0 versus 4I0).

A thickened perichondrium could be the result of an increasein matrix deposition (perhaps a kind of scar tissue). Alternatively,the thickening could represent an increase in the number of cells.These cells could be predominantly vascular, providing asupportive niche; alternatively, the cells could be chondrogenic

Fig. 4. Costal cartilage repair. (A, A0) When both the cartilage and perichondrium were removed, no repair occurs even at �9 months (269 days) post‐resection (PR). (B, B0) When the cartilage is removed while the perichondrium is retained in the animal, cartilage spans the resection zone andbecomes mineralized, �9 months (269 days) PR (A, B, skeletal preparation, A0–B0, mCT, CD‐1 mice, thin blue lines in the resected region are sutures). (C)CD‐1 resection in which the perichondrium is retained in the animal, stained with alcian blue and alizarin red, 1 week PR showing alcian blue–positivestaining near the cut ends. (D) After 3 weeks PR, alcian blue staining could be found spanning the resection, CD‐1. (E) Sample at 9 months (274 days)PR showing cartilage repair across the resected region, alizarin red/alcian blue, CD‐1. Arrowheads mark the resection boundaries; dashed lines indicatethe position of serial sections. (F) Section through normal rib stained with hematoxylin and eosin at location indicated in E. Although the surroundingtissue was disrupted by the skeletal preparation, the cartilage was relatively intact. (G1–G3) Sections through the resected region at locationsindicated in E. (H, I). Another CD‐1 sample with a similar PR time (�9 months, 279 days) but not subjected to skeletal preparation to preserve thehistology, stained with hematoxylin and eosin. H, H0 are through normal, unresected rib. I, I0 are through a resected rib. (H0, I0) The perichondrium athigher magnification. (J, K) PR healing of �1 month (31 days), Gomori’s trichrome, shown at high and low magnification. (L, M) A cross section throughunresected rib and resected rib (2 weeks PR), respectively, stained with hematoxylin and eosin. (L0) A near adjacent serial section to L with cells weaklypositive for PCNA in the perichondrium and at the cartilage periphery. (M0) A near adjacent serial section to M with high PCNA expression in the repairregion including the greatly thickened perichondrium. (A–E) Scale bar¼ 1mm, (F–K) scale bar¼ 100 microns, (H0 , I0, J0, K0 , L, L0, M, M0) scale bar¼ 50microns. All micrographs: minor adjustment with Photoshop Levels to correct for underexposure.

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Fig. 4.

302 SROUR ET AL. Journal of Bone and Mineral Research

progenitors that expand in response to injury for the purpose ofmediating repair. In support of this latter hypothesis, a thickenedperichondrium (1.98 times thicker) was also evident at earlier PRtime points (compare Fig. 4J0–K0) and within the expanded zone,abundant cells were evident with prehypertrophic profiles inisogenous groups characteristic of chondrogenesis. Further-more, cells in this location expressed PCNA and, therefore, wereactively proliferating (compare Fig. 4L0 to Fig. 4M0). Theseobservations suggest that an important response to injuryinvolves the expansion of progenitors within the perichondriumand that these progenitors maymediate repair by differentiatinginto cartilage and filling in the resected region. After the initialthickening, the perichondrium maintains its thicker dimensions,potentially housing now quiescent progenitors.The new cartilage underwentmineralization (like flanking costal

cartilages had during postnatal development) as evidenced by

alizarin red staining (Fig. 4B, E and Fig. 5A, B). Specimens with longhealing times had resections that were eventually completelymineralized (Fig. 4B, B0). In addition, we could usemineralization asa proxy for the extent of repair and measured mineralizationvolume with mCT imaging in both wild‐type and MRL/MpJspecimens (a random subset of the cohort was analyzed, 80 to293 days PR healing times,n¼ 28). To quantify the extent of repair,we used measurements from reconstructed mCT scans (examplesshown in Fig. 4A0, B0) to develop a mathematical model that tookinto consideration healing via extension from the ends (apposi-tionally) and/or filling in (as though from progenitors located inthe perichondrial sleeve) and incorporated a Bayesian statisticalfitting procedure to account for sources of variation anduncertainty including measurement error, model misfit, andrandom variability between animals. Using this approach, wefound that although there may be differences early in the healing

Fig. 5. Quantification of costal cartilage repair. (A, B) Alizarin red/alcian blue panels are shown on the left for cartilage‐only removal experiments inrepresentative MRL and CD‐1 animals at �3 months PR. (A0, B0) Equivalent views of the same samples as in A and B from mCT scan reconstructionsshowing the quantified masked region (red). (C) Model showing mineralization over time and demonstrating that in both CD‐1 (black) and MRL (green)mouse strains, the same healing process is occurring during the time points analyzed. To understand the scales of the axes, see Materials and Methods.Because volume is proportional to r2, the increased asymptotic volume of between 1.5 and 3� corresponds to only a modest thickening of the rib of�20% to 70%. (D) Graph showing the relationship between the surface‐to‐volume ratio, or “thinness” and the percent healing/time, or “amount ofrepair.” Line shows a linear regression trend line with a slope of 516.3. The Pearson correlation coefficient was calculated to be 0.479 (p¼ 0.009); regionswith low volume and high surface area (long, thin regions) differentiatedmore completely. Scale bars¼ 1mm. (A–B0) All micrographs: minor adjustmentwith Photoshop Levels to correct for underexposure.

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process, both wild‐type and MRL/MpJ strains mineralizedasymptotically to a similar degree, and they did so on a time‐scale related to the geometric properties of the rib resection,especially the surface area to volume ratio, suggesting that fillingin from the periphery was the main repair mechanism (Fig. 5C).Interestingly, although in some cases the repair could have asomewhat larger volume than the resected region, the newcartilage consistently maintained a cylindrical profile and did notexhibit extensive protuberances or a grossly enlarged callus,suggesting that either the perichondrium constrained the repairor that another unknown mechanism regulates cartilage shapeand size.

The perichondrium clearly plays a critical role in facilitatingrepair because removal of both the cartilage and the surround-ing perichondrium failed to support cartilage regrowth evenafter �9 months PR (Fig. 4A, A0). In addition, in our “cartilageonly” removal experiments, we found a strong correlation(Pearson correlation coefficient¼ 0.491, p¼ 0.008) between theamount of repair (% of area filled in, based on time) and the“thinness” (surface area/volume ratio) of the resected region,with thin, large surface area to small volume resectionsexhibiting the most complete degree of repair (Fig. 5D). Theseresults suggest that the fastest repair occurs when the resectedregion has a small volume and is surrounded by abundantperichondrium.

The ability of cells to retain a DNA label has been used toidentify slow‐cycling cell populations that in some contexts—hair follicle and bone marrow and auricular cartilage—correlatewith the location of resident stem cells.(14,22,23) To determinethe location of label‐retaining cells in costal cartilages, wepulse‐chase labeled animals with EdU(24) starting at postnatalday 10. After a short chase (8 hours), very few cells were labeledin both the perichondrium and mature cartilage (data notshown). However, after a 14‐day and 8‐month chase, labeledcells could be found both within the cartilage matrix andperichondrium (Fig. 6A–C0). After 14 days, labeled cells withinthe cartilagematrix were clustered in small groups of 2 to 3 cellsand located predominantly at the periphery (Fig. 6A, B, greenarrows), although occasionally small clusters could also befound in the cartilage interior (not shown). The lack of labeledcells within the cartilage after a short chase and the smallclusters found after a long chase suggests that new chon-drocytes form preferentially from perichondrium progenitors atleast at these postnatal stages. Even after an 8‐month chase,rare label‐retaining cells could be found in the perichondriumand occasionally presumably new chondrocytes (pairs indicat-ed with green arrows) could be seen in the periphery (Fig. 6C,C0). The perichondrium has been shown to be composed ofdistinct layers (inner and outer), which are hypothesized to havedistinct functions (inner, appositional growth at the periphery;outer layer structural and vascular support) and have classicallybeen visualized with light or electron microscopy but recentlyshown to express specific molecular markers.(15,25) Labeled cellsin the perichondrium were counted and classified as inner orouter cells (Fig. 6B, B0). For the most part, classification wasunambiguous as labeled cells were typically found either at theinner or outer extremes, suggesting that the perichondrium hasdistinct populations of cells that are not highly proliferative. Ashave been found in other contexts,(14) we did find anenrichment of label‐retaining cells within the perichondrium;however, after the 14‐day chase, we found more labeled cells inthe outer than inner layer (2.2 times more, p¼ 0.018, paired ttest), whereas after 8 months, labeled cells could only be found

within the inner layer. Although it is possible that labelretention represents cells that only divided once or twice andthen differentiated, it is also possible that the label‐retainingcells represent stem cells. Further lineage analysis will beneeded to determine this more precisely.

We next tested the capacity of the perichondrium to supportnew cartilage generation by placing the perichondrium in anectopic location (Fig. 6D). These strips were largely devoid ofalizarin red staining, indicating that our dissection technique didnot carry along large pieces of adherent mature cartilage (Fig. 6E).We then chose a nearby intercostal muscle, created a glancingflap, and sutured the perichondrium inside. By visual inspection,we observed no evidence of graft resorption or extrusion at 1, 2,and 3 weeks after surgery. We then assayed for the formation ofdifferentiated cartilage with alizarin red staining. We found thatregardless of the healing time (ranging from 2weeks to 5months)ectopic mature cartilage was evident (Fig. 6F). This ectopiccartilage could take up calcein (Fig. 6G), indicating newmineralization. Samples analyzed up to 2 months had largecartilage pieces, but by 5 months only small fragments werepresent (data not shown), suggesting that the generation andretention of cartilage in this location was robust (n¼ 10/15) butlimited over periods 2 months and longer. A tdTomato‐labeledperichondrium implantation also resulted in ectopic cartilage.Both mature and prehypertrophic cartilage cells were tdTomatopositive, indicating that the ectopic cartilage arose from donorrather than host tissue (Fig. 6H, H0). Furthermore, the ectopic tissuewas proliferative, as evidenced by PCNA expression (Fig. 6I, I0).

Discussion

Time sequence of repair

In humans, regeneration of the costal cartilage has beenobserved but not rigorously documented or analyzed at thecellular level. Here, we establish a simple baseline model in amammal that allows further studies of cartilage repair toproceed within the context of the enormous flexibility and lowcost of the mouse genetic system. From this initial study, wepropose a timeline for perichondrium‐mediated cartilage repairover the course of 1 to 2 months (Fig. 7).

Within the first week, chondrogenic progenitors within theperichondrium expand. Next, we propose that these progen-itors fill the resection zone by emerging both from theperichondrium lateral to the cut site and from the remainingperichondrium along the length of the resection. Newchondrogenic progenitors proliferate to create isogenousgroups, secrete cartilage matrix, and differentiate into maturechondrocytes with increasing hypertrophic profiles. Althoughthe cartilage in the repair zone contains chondrocytes withvariable hypertrophy and tends to be more nodular thannormal (Fig. 4I) (how chondrocyte size and, therefore, cartilagesize may be regulated is still to be fully determined(26)), theregenerated cartilage fully differentiates, producing a mineral-ized matrix as is typical of mouse costal cartilage. Thus, as infracture repair where the surrounding connective tissue(periosteum) has been shown to play an important role,(12,27)

cartilage repair in this context may also be mediated by cellularcontributions from the surrounding connective tissue (peri-chondrium). Furthermore, the sequence of events observedhere likely recapitulates some of the steps that occurred duringdevelopment (expansion of progenitors, increased hypertro-phy, mineralization).(28)

304 SROUR ET AL. Journal of Bone and Mineral Research

Use of mice

More generally, the establishment of this mouse model allowsfor an enormous variety of genetic, drug, and surgicalinterventions to be compared within transgenic and genetically

altered backgrounds. For example, in this study, we compare therepair capabilities of both wild‐type and the mouse strain MRL/MpJ. Although we do not see an enhanced rate of repair in thisbackground within the time frame examined (months), theremay be differences in the repair rate within the first 2 to 4 weeks.

Fig. 6. The perichondrium contains slow‐cycling cells and can support new cartilage growth. (A–A00) Careful observation was made to distinguish cellslabeled with EdU (green arrows) from autofluorescing red blood cells (yellow arrows), which are smaller, rounder, largely found within capillaries, andwere Hoechst negative. (B) EdU/Hoechst double‐positive perichondrium cells (white arrows, arrowheads) could be distinguished from maturechondrocytes (green arrows) and were counted as inner (white arrows) or outer (white arrowheads) based on location and Gomori’s trichrome staining(B0). (C, C0) Rare EdU‐positive cells could be located in animals with an 8‐month chase located within the inner layer only. Label‐retaining chondrocytes(presumably new) could sometimes be found at the cartilage periphery. (D) Diagram showing how the perichondrium (gray) was stripped from the costalcartilage and grafted into the center of a nearby intercostal muscle. (E) A perichondrial strip stained with alizarin red/alcian blue. Some cartilage didadhere, but the majority was removed. (F) New cartilage development was evident at 2 weeks as demonstrated by alizarin red staining. (G) Newmineralization as indicated by calcein dye incorporation (inset shows brightfield image). (H, H0) When a labeled donor is used, ectopic cartilage istdTomato‐positive and contains mature and prehypertrophic cartilage in isogenous groups (yellow and white arrowhead, 2 weeks postimplantation) (His stained with hematoxylin and eosin; H0 is the same section in fluorescence). (I, I0) The implant contained cells positive for PCNA expression (I is stainedwith hematoxylin and eosin; I0 is a near adjacent section in fluorescence; cells negative for PCNA are indicated with arrowheads). (A–C, H, H0, I, I0) Scalebar¼ 50 microns; (C0) scale bar¼ 25 microns; (E, F) scale bar¼ 1mm; (G) scale bar¼ 100 microns. All micrographs: minor adjustment with PhotoshopLevels to correct for underexposure.

Journal of Bone and Mineral Research RIB CARTILAGE REGENERATION IN A MOUSE MODEL 305

Future studies will involve characterizing the early steps ofcartilage repair to understand the cellular dynamics (infillingversus appositional growth) and examining repair within thecontext of other loss‐of‐function or gain‐of‐function alleles,particularly for cartilage‐inducing growth factors. Through thesestudies, themolecular nature of the repair competency of the ribcould be elucidated.

Perichondrial progenitors

During development, the perichondrium is a major source ofchondrogenic and osteogenic progenitors.(27,29,30) During carti-lage repair, the perichondrium may be a critical player andrepresents an important area of interest for developing newtherapies. For example, in the intervertebral disc, the location oflabel‐retaining, slow‐cycling cells as well as the expression ofstem cell markers suggests the presence of a niche in theperichondrium adjacent to the edge of the annulus fibrosus.(8) Inaddition, similar studies in the knee joint have suggested that aniche might exist in a similar perichondrium‐like region, calledthe groove of Ranvier,(31) or in the articular surface layer, whichmay have remnant characteristics of the perichondrium.(9,32)

Label‐retaining cells have been found in the perichondriumsurrounding the elastic cartilage of the mouse auricle, andperichondrial cells have been isolated from mouse and humanauricle that have chondrogenic capacity.(14,33) In this study, we alsofind label‐retaining cells within both the inner and outer layers ofthe perichondrium after a 2‐week chase and within the inner layerafter an 8‐month chase (Fig. 6A–C). Furthermore, the ectopiccartilage found after intramuscular perichondrium implantationcontains bothmature and newprehypertrophic cartilage cells andis derived from the donor tissue (Fig. 6E–I0), providing further

support for the idea the perichondrium is the source of cells forrepair rather than simply providing a scaffold for chondroproge-nitors derived from some other location. Future studies usingtechniques to both isolate perichondrial cells and observe theirpotential in culture and to track their lineage in response to injuryare needed. Fortunately, genetically modified mouse lines havebeen successfully employed to address similar questions in theepidermis, and this technology could be reapplied here.(34)

General use of rib‐derived or “riblike” cells in cartilagerepair

Because of morbidity problems with harvesting autologouscartilage for grafting purposes, there is great interest in cell‐based therapies to repair damaged cartilage. Currently, themost common approach is to culture and transplant autolo-gous articular chondrocytes for acute joint injuries repair.(35)

Unfortunately, these cells can have limited viability, fail toproliferate, may undergo dedifferentiation, or even fail tointegrate.(36) Another potential source for cells is costalcartilage, and this source has some advantages. Costalcartilage is a hyaline‐type cartilage similar to the cartilagefound in the articular region. Chondrocytes can be readilyprocured from the patient and may expand more efficiently invitro than articular cartilage. Furthermore, costal cartilage thathas dedifferentiated in culture can be converted into high‐quality hyaline cartilage.(37) In addition, the use of costalcartilage cells has been shown to be a feasible option forarticular cartilage repair in animal models.(38,39) However,costal cartilage as well as cartilage generated from othersources can have a tendency to undergo mineralization overtime. For the purpose of articular cartilage defect repair, thismay be good for reconstructing the osteochondral junction sothat the articular surface does not delaminate. However, thismay present challenges for articular surface reconstruction,which needs to remain unmineralized for best function.

Mineralization poses less of a problem for the use of costalcartilage in craniofacial and trachea repair. Thus, the use ofcostal cartilage and/or the use of banked stem cells derivedfrom costal cartilage perichondrium may be an attractiveoption for various cartilage‐ or even bone‐repair applications.A handful of studies have demonstrated a robust ability ofrib perichondrium to facilitate the repair of articular defectsin rabbits and sheep.(40,41) Given our findings, it may bebeneficial to revisit these injury studies using genetictechniques to track and lineage‐trace implanted cells.Furthermore, developing efficient ways to isolate chondro-cyte progenitors from rib perichondrium may have highlyuseful clinical applications.

The embryonic origin of the rib is distinct from that of theappendicular skeleton (somite versus lateral plate meso-derm(42)). Thus, one possible reason for the high regenerativecapacity of ribs may be related to the developmental historyexperienced by rib versus limb progenitor cells. One idea wouldbe to mimic this history by transitioning generic pluripotentcells through a somite mesoderm‐like induction process. Forexample, Nakayama and colleagues have shown that humanpluripotent cells transitioned in this way have enhancedchondrogenic capacity when compared with human bonemarrow–derived mesenchymal stem cells,(43) and it will beinteresting to determine if these cells share a commonmolecular profile with rib‐derived progenitors. Althoughbanking perichondrium‐derived cells may be a feasible option,

Fig. 7. In this model, the perichondrium supports new cartilage duringrepair. (A) Costal cartilage is mineralized at the time of surgery (darkgray). When the perichondrium is retained in the animal (dark gray),repair along the length of the excised region is proposed to bemediatedby perichondrial‐derived progenitors from the ends and the remainingperichondrium (black arrows). (B) New cartilage completely fills in theregion (patterned cylinder). (C) These chondrocytes eventually differen-tiate to produce a mineralized matrix (dark gray regions), and the entireresection zone eventually fills in dark gray.

306 SROUR ET AL. Journal of Bone and Mineral Research

riblike chondrogenic progenitors generated from inducedpluripotent cells may be easier to develop for clinical use. Ineither case, given our findings, it is attractive to imagine usingthese rib or riblike progenitors in a variety of applications,including acute cartilage injury repair, auricle and tracheareconstruction, rhinoplasty, and even myringoplasy,(44,45) andwe hope future studies will investigate these possibilities.In conclusion, here we present a simple method for the study

of large segmental cartilage defects in a mammalian model.Similar to sporadic reports in humans (Kawanabe and Nagata,(6)

Lester,(7) Chang and colleagues,(11) and this study, Fig. 2), we findthat themouse costal cartilage repairs well, within 1 to 2months,and we have identified the perichondrium as a key player andthe likely source of repair cells.

Disclosures

All authors state that they have no conflicts of interest.

Acknowledgments

We thank Sara Sabet for technical assistance and the GriksheitLab for sharing reagents. The clinical study was partiallysupported by an Oral & Maxillofacial Surgery FoundationResearch Award (JSL). Our other funding sources were: theBaxter Medical Scholar Research Fellowship (MKS and KTY), USCundergraduate fellowships (MKS and SL), and the Provost, DeanJoan M. Schaeffer, and Rose Hills Fellowships (MKS). We alsoacknowledge a California Institute of Regenerative Medicine(CIRM) training fellowship (JSL), a CIRM BRIDGES fellowshipthrough California State University, Fullerton (APM) andPasadena City College (AKI), and the James H. ZumbergeResearch and Innovation Fund, the USC Regenerative MedicineInitiative, and the NIAMS NIH under Award no. R21AR064462 toFVM.Authors’ roles: MKS, KTY, JLF, and FVM designed the

experiments. MMU assisted with the surgical procedures design.JSL performed the case study. MKS, JLF, KY, APM, AI, ALM, and SLperformed and analyzed experiments. DLL contributed therepair rate model. All authors contributed to the interpretationof results. MKS and FVM wrote the manuscript, and all authorscontributed to the editing of the manuscript.

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