Cellular therapy in bone-tendon interface regeneration

Figures & data

Figure 1. Histological features of the fibrocartilaginous bone-tendon interface stained with (A) Safranin-O, (B) H&E, and viewed with (C) polarized light microscopy. Note the four zones of the native enthesis. Magnification 20×. B, bone; CFC, calcified fibrocartilage; UFC, uncalcified fibrocartilage; T, tendon. Reproduced with permission from reference 122.

Figure 2. Embryonic development of the bone-tendon interface. (A) The degree of Scx and Sox9 expression determines cellular phenotype. Scx⁻/Sox9⁺ chondroprogenitors (CP) become chondrocytes of the skeletal anlagen while Scx⁺/Sox9⁻ tenoprogenitors (TP) become tenocytes of the tendon midsubstance. Varying levels of Scx and Sox9 expression are seen in teno-/ligamento-/chondro-progenitors (TLCP), which give rise to cells of the bone-tendon interface. (B) Scx⁺/Sox9⁺ progenitors give rise to the primordial chondro-tendinous/ligamentous junction (CTJ/CLJ), which forms the osteo-tendinous/ligamentous junction (OTJ/OLJ) following birth. Reproduced with permission from reference 23.

Figure 3. Cell sources for augmenting bone-tendon healing. Animal studies exploring the use of MSCs to improve bone-tendon healing have derived these cells primarily from the bone marrow, although other tissues (e.g., adipose) also harbor MSCs (not shown). ACL-derived MSCs can be obtained from the ruptured ligament, while chondrocytes/cartilage plugs can be taken from non-weightbearing articular surfaces. Lastly, the periosteum is typically harvested from the anteromedial tibia, given the ease of access in the absence of overlying soft tissues.

Table 1. In vivo studies of cellular therapy in bone-tendon interface healing

Cell Source	Site of Repair	Animal Model	Results	Ref.
Anterior Cruciate Ligament (ACL) Tissue/Cells
Autologous ruptured ACL	Anterior cruciate ligament	• Beagle dogs • Bilateral ACL reconstruction with ipsilateral flexor digitorum superficialis tendon autograft • “Tissue-treated” group augmented with resected native ACL tissue in tibial bone tunnel	• Tissue-treated group showed endochondral ossification-like integration with enhanced angiogenesis and osteogenesis starting at 2 wk • At week 4, Tissue-treated group had smaller tibial tunnel cross-sectional area as compared with contralateral control • At week 4, ultimate load in tissue-treated group was over twice that of controls 80% of controls failed at bone tunnel; Only 20% of tissue-treated samples failed at tunnel	69
ACL-derived stem cells	Anterior cruciate ligament	• Nude rats • ACL reconstruction with intracapsular injection of 5x10^5 human stem cells obtained from ruptured ACL • Four experimental groups – (1) CD34+ cells, (2) Nonsorted cells, (3) CD34- cells, (4) no cells	• Human stem cells honed to healing bone-tendon interface • Collagen fiber deposition and collagen 2-expressing cells seen at 2 wk • Human stem cells induced angiogenesis and osteogenesis in rat cells, and differentiated into these lineages themselves • Bone volume/total tissue volume increased at 4 wk in CD34+ and NS groups • **In all above cases, greatest effects seen in CD34+ > NS > CD34- cells • At week 8, ultimate load greatest in CD34+ > NS > CD34- cells	70
ACL-derived stem cell sheets	Anterior cruciate ligament	• Nude rats • ACL reconstruction with flexor digitorum longus autograft • Three experimental groups – (1) autograft wrapped with human CD34+ cell sheet, (2) Intra-articular injection of CD34^+ cells, (3) no cells	• Transplanted cells observed directly in tendon graft and at bone tunnel; Greater number for cell sheet vs intra-articular injection • Increased vascularization and collagen type 3 expression in tendon graft • At two weeks, evidence of intrinsic osteogenesis and neovascularization around bone tunnel; Human cell markers also co-localized with endothelial and osteogenic markers • ** In all above cases, greatest effects seen in sheet > injection > no cells • At week 8, ultimate load greatest in sheet > injection > no cells	71
Chondrocytes/Cartilage
Allogeneic chondrocyte pellet	Partial patellectomy	• NZW rabbit • Distal 1/3 patella resection • Placed chondrocyte pellet between remaining patella and ligament • Immobilized 4 wk long cast	• Chondrocyte pellet placed between 1/3 distal-resected patella and patella ligament showed improved integration and formation of fibrocartilge-like zone at weeks 12 and 16. No integration seen without pellet • No mechanical testing performed • Collagen/mineral composition of interface not performed	72
Articular cartilage plug	Partial patellectomy	• Chinese goats • Distal 1/3 patella resected and patella tendon sutured to proximal 2/3 patella • Repair alone vs. cartilage wafer (from autologous hyaline cartilage on resected 1/3 patella • Casted 6 wk, weight-bearing immediately	• Autologous articular cartilage interposition resulted in more fibrocartilage transition zone formation than direct repair at all times • No group differences in new bone formation, but increased with time in both groups • Ultimate load increased for both groups, while ultimate stress did not change. No group differences at any time	73
MSCs or Chondrocytes in fibrin clot	Achilles	• Male Wistar rat • Achilles enthesis destroyed with transection and burr • Repair with suture through bone tunnels • Three experimental groups (1) repair only, (2) 4x10^6 chondrocytes in 100 ul fibrin clot, (3) 4x10^6 MSCs in 100 ul fibrin	• Both chondrocytes and MSCs produced fibrocartilage (as demonstrated by Col2 and GAG staining) starting at day 15. No fibrocartilage in untreated group up to 45 d • On day 45, only MSC recapitulated columnar structure of hypertrophic chondrocytes • Failure to load improved over time for all groups, but chondrocyte and MSC significantly higher than controls at day 45 (and equal to native controls)	74
Enthesis Cells
Tendon-to-bone interface cells	Supraspinatus	• Lewis Rats • 2x2 mm of supraspinatus excised and enthesis destroyed Five groups – (1) native control, (2) no repair, (3) suture repair, (4) suture repair with gelfoam, (5) suture repair with gelfoam + 1.5 x 10^5 cells	• At week 3, gelfoam + cell group (V) displayed increased cellularity, vascularity, inflammation, and collagen disorganization vs. other groups • At week 12, gelfoam + cell group (V) had better collagen organization than other treatment groups (II-IV) • Cells retained at healing site up to 12 wk No biomechanical testing performed	75
Mesenchymal Stem Cells (MSCs)
BMSCs in fibrin clot	Supraspinatus	• Lewis rats • Supraspinatus transection with acute repair augmented with nothing, fibrin clot, or fibrin + allogeneic BMSCs • Not immobilized	• Ad-LacZ-transduced MSCs demonstrated cell viability at 2 and 4 wk (but decreasing) • No difference in amount of new cartilage or collagen fiber organization between groups at either time point • Mechanical properties improved with time, but no group differences	83
MT1-MMP-transduced MSCs in fibrin clot	Supraspinatus	• Same methodology as^83 • MSCs in fibrin clot vs. MT1-MMP-transduced MSCs at weeks 2 and 4	• Greater chondrogenesis (Safrinin O staining) in MT1-MMP-transduced MSCs at week 4, as compared with MSC alone. No difference at week 2. • No group difference in collagen alignment (picrosirius red) at either time point • Mechanical properties greater in MT1-MMP MSCs than MSCs alone at week 4 only	88
BMP13-transduced MSCs in fibrin clot	Supraspinatus	• Same methodology as [2] • MSCs in fibrin clot vs. BMP13-transduced MSCs at weeks 2 and 4	• Improved biomechanical properties over time, but no group differences • No group differences in histology (safranin O for fibrocartilage formation and polarized picrosirius red for collagen organization)	84
Scx-transduced MSC in fibrin clot	Supraspinatus	• Same methodology as^83^,^88 • MSCs in fibrin clot vs. Scx-transduced MSCs at weeks 2 and 4	• Ad-Scx MSCs improved mechanical properties at 2 and 4 wk, but absolute values not clearly better than MT1-MMP-mediated improvements [3] • Increased fibrocartilage formation (as determined by Safranin O staining) in Scx-MSC group, but no group differences in collagen organization	87
BMSCs on poly-glycolic scaffold	Infraspinatus	• Japanese White Rabbit • Full-thickness (10x5mm) defect made in infraspinatus tendon • Defect filled with (1) nothing (control), (2) PGA scaffold, or (3) PGA scaffold seeded with 5x10^6 cells	• Left untreated, defect healed with poorly organized fibrovascular scar, with no improvements in mechanical properties from 4 to 16 wk • At 4 wk, both PGA and PGA+MSC groups showed partially degraded scaffold with foreign body reaction and multinuclear cell infiltrate • At week 8, chondrocytes seen in PGA+MSC group only, but at week 16, both groups showed fibrocartilage formation and positive Safranin-O staining • PGA group showed greater Collagen 3 to Collagen 1 staining at weeks 8 and 16; Conversely, PGA+MSC group showed Col1 > Col3 staining at week 16 • No group differences in biomechanical properties at week 4, but by week 16 PGA+MSC > PGA > > > controls (defect only)	93
MSCs in fibrin coating allograft	Anterior cruciate ligament	• NZW rabbit • Bilateral ACL reconstruction with Achilles tendon allograft • Graft coated with (1) fibrin or (2) fibrin + 4 × 10^6 BMSCs	• Fibrin group showed fibrovascular scar in interface at weeks 2, 4, 8 • MSC group showed chondrocyte-like cells in interface at 2 wk; by week 8, differentiated enthesis with 4 zone transition; Copious GAG and Col2 staining • At 8 wk, load to failure higher in MSC than controls. No significant groups differences at earlier time points, or in stress, stiffness, Young’s modulus • Major mode of failure was graft pullout (wk 2) and midsubstance rupture (wk 8); no group difference	82
BMSCs in fibrin sealant coating tendon allograft	Anterior Cruciate ligament	• NZW Rabbits • Bilateral ACL reconstruction with hamstring allograft, coated with (1) fibrin or (2) fibrin + BMSCs	• Fibrin group showed fibrovascular interface at weeks 2, 4, 8 • Fibrin + MSC group showed disorganized chondrocytes at week 2. Increasing fibrocartilage organization at weeks 4 and nearing native enthesis histology at week 8, with graded fibrocartiaginous interface • Load to failure and stiffness did not change over time in fibrin controls; Both increased over time in MSC group, with significant group differences at week 8	80
Synovial MSCs	Medial patella tendon reconstruction	• Sprague-Dawley rats • Medial patella tendon resected and replaced with ipsilateral ½ Achilles tendon autograft; Proximal graft sutured to patella periosteum and distal portion inserted into bone tunnel from tibial plateau to tibial tuberosity • Bone tunnel injected with (1) collagen gel (control) or (2) collagen gel + 1x10^7 synovial MSCs	• At week 1, both control and MSC-group showed fibrovascular scar in interface • By week 4, implanted tendon attached directly to bone, with no observable fibrocartilage in either group • At week 4, MSC-group had higher percentage of oblique (i.e., Sharpey) fibers relative to total interface area, as compared with controls • No biomechanical testing	120
Murine mesenchymal cell line (C3H10T[1/2] and human BMSC virally-mediated overexpression of Smad8ca and BMP2	Heterotopic (intramuscular)	• Nude mouse • Intramuscular injection of murine C3H10T[1/2] mesenchymal cell line or human BMSC with virus-mediated overexpression of constitutively active Smad8 (Smad8ca) and BMP2	• At 1 mo, C3H10T[1/2] cells with Smad8ca and BMP2 overexpression formed bone ossicle and tendon-like structure with interposing fibrocartilage-like structure • Murine cells infected with Smad8ca alone produced tendon-like tissue • Human BMSCs with lentiviral-mediated overexpression of Smad8ca and BMP2 formed bone ossicle and tendon-like structure with no intervening fibrocartilage • Neither uninfected cells nor wild-type Smad8 expression produced neotissue	121
BMSC	Heterotopic (Hallucis longus tendon in calcaneal bone tunnel)	• NZW rabbit • Hallucis longus tendon inserted into calcaneal bone tunnel • Augmented with (1) fibrin gel (control) or (2) fibrin gel + 1 × 10^7 BMSC	• Control group showed increasing organization of fibrovascular scar at 2, 4, 6 wk • BMSC group showed fibrocartilage formation over 4 wk, but only at 50% of bone-tendon interface • Both groups stained positive for Collagen 1 and 3; Only BMSC group stained positive for collagen 2 • No mechanical testing	81
Periosteum/Periosteum progenitor cells
Autologous periosteum or bone marrow aspirate	Heterotopic (extensor digitorum longus tendon in bone tunnel)	• NZW rabbits • Femoral insertion of extensor digitorum longus resected and inserted in tibial bone tunnel • (1) non-augmented (C) and (2) wrapped with periosteum (P), or (3) bone marrow aspirate (BMA)	• P and BMA groups showed increased interface organization as compared with controls at both weeks 6, 12 • Chondrocyte-like cells seen in BMA group at week 6; Fibrocartilage islands seen in both BMA and P group at week 12; both stained positive for Safranin O • At week 6, load to failure and stiffness P > BMA > C; At week 12, load to failure significantly increased over controls in P alone, with stiffness statistically equivalent across groups	96
Autologous periosteum	Heterotopic (extensor digitorum longus tendon in bone tunnel)	• NZW rabbits • Femoral insertion of extensor digitorum longus resected and inserted in tibial bone tunnel • (1) non-augmented (control) and (2) wrapped with periosteum	• For periosteum-treated group, fibrovascular scar seen at 4 wk; At week 8, maturation of new bone growing into fibrous tissue, with increasing mineralization; At 12 wk, fibrocartilage formation identified; No histology for control groups was shown or described • Load to failure increased over time • Load to failure significantly higher in periosteum-treated group than controls at weeks 8 and 12	97
Autologous periosteum	Infraspinatus	• NZW rabbits • Acute transection of infraspinatus and footprint debridement • Transosseus repair with or without autologous tibial periosteum interposed between tendon and bone	• Both control and periosteum-treated groups showed healing of infraspinatus to humerus • Periosteum-treated group showed fibrovascular scar in interface at week 4, chondrocyte-like cells at week 8, and anatomy similar to native enthesis with tidemark at week 12; No histology for control group was shown • Both groups showed increased load to failure over time; Periosteum-treated group significantly greater than controls at weeks 8 and 12	98
Periosteum progenitor cells with BMP2-tethered hydrogel	Infraspinatus	• NZW white rabbit • Infraspinatus transected and enthesis destroyed • Transosseus repair with interposed hydrogel; 3 groups – (1) native control, (2) PEGDA hydrogel, (3) PEGDA hydrogel + BMP2 + PPCs	• At weeks 4 and 8, both groups demonstrated increasing fibrocartilage formation • Hydrogel containing BMP2 and PPCs demonstrated improved fibrocartilage and bone formation determined by histology, as compared with hydrogel alone • Ultimate load increased over time in both groups, but was higher in PPC groups at both weeks 4 and 8	99

Lui PPY, Zhang P, Chan KM, Qin L. Biology and augmentation of tendon-bone insertion repair. J Orthop Surg Res 2010; 5 14; http://dx.doi.org/10.1186/1749-799X-5-59; PMID: 20727196

 PubMedGoogle Scholar

Sugimoto Y, Takimoto A, Akiyama H, Kist R, Scherer G, Nakamura T, Hiraki Y, Shukunami C. Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament. Development 2013; 140:2280 - 8; http://dx.doi.org/10.1242/dev.096354; PMID: 23615282

 PubMed Web of Science ®Google Scholar

Matsumoto T, Kubo S, Sasaki K, Kawakami Y, Oka S, Sasaki H, Takayama K, Tei K, Matsushita T, Mifune Y, et al. Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue. Am J Sports Med 2012; 40:1296 - 302; http://dx.doi.org/10.1177/0363546512439026; PMID: 22427618

 PubMed Web of Science ®Google Scholar

Mifune Y, Matsumoto T, Ota S, Nishimori M, Usas A, Kopf S, Kuroda R, Kurosaka M, Fu FH, Huard J. Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. Cell Transplant 2012; 21:1651 - 65; http://dx.doi.org/10.3727/096368912X647234; PMID: 22732227

 PubMed Web of Science ®Google Scholar

Mifune Y, Matsumoto T, Takayama K, Terada S, Sekiya N, Kuroda R, Kurosaka M, Fu FH, Huard J. Tendon graft revitalization using adult anterior cruciate ligament (ACL)-derived CD34+ cell sheets for ACL reconstruction. Biomaterials 2013; 34:5476 - 87; http://dx.doi.org/10.1016/j.biomaterials.2013.04.013; PMID: 23632324

 PubMed Web of Science ®Google Scholar

Wong MWN, Qin L, Tai JKO, Lee SKM, Leung KS, Chan KM. Engineered allogeneic chondrocyte pellet for reconstruction of fibrocartilage zone at bone-tendon junction--a preliminary histological observation. J Biomed Mater Res B Appl Biomater 2004; 70:362 - 7; PMID: 15264320

 PubMed Web of Science ®Google Scholar

Wong MWN, Qin L, Lee KM, Leung KS. Articular cartilage increases transition zone regeneration in bone-tendon junction healing. Clin Orthop Relat Res 2009; 467:1092 - 100; http://dx.doi.org/10.1007/s11999-008-0606-8; PMID: 18987921

 PubMed Web of Science ®Google Scholar

Nourissat G, Diop A, Maurel N, Salvat C, Dumont S, Pigenet A, Gosset M, Houard X, Berenbaum F. Mesenchymal stem cell therapy regenerates the native bone-tendon junction after surgical repair in a degenerative rat model. PLoS One 2010; 5:e12248; http://dx.doi.org/10.1371/journal.pone.0012248; PMID: 20805884

 PubMedGoogle Scholar

Loeffler BJ, Scannell BP, Peindl RD, Connor P, Davis DE, Hoelscher GL, Norton HJ, Hanley EN Jr., Gruber HE. Cell-based tissue engineering augments tendon-to-bone healing in a rat supraspinatus model. J Orthop Res 2013; 31:407 - 12; http://dx.doi.org/10.1002/jor.22234; PMID: 23070709

 PubMed Web of Science ®Google Scholar

Gulotta LV, Kovacevic D, Ehteshami JR, Dagher E, Packer JD, Rodeo SA. Application of bone marrow-derived mesenchymal stem cells in a rotator cuff repair model. Am J Sports Med 2009; 37:2126 - 33; http://dx.doi.org/10.1177/0363546509339582; PMID: 19684297

 PubMed Web of Science ®Google Scholar

Gulotta LV, Kovacevic D, Montgomery S, Ehteshami JR, Packer JD, Rodeo SA. Stem cells genetically modified with the developmental gene MT1-MMP improve regeneration of the supraspinatus tendon-to-bone insertion site. Am J Sports Med 2010; 38:1429 - 37; http://dx.doi.org/10.1177/0363546510361235; PMID: 20400753

 PubMed Web of Science ®Google Scholar

Gulotta LV, Kovacevic D, Packer JD, Ehteshami JR, Rodeo SA. Adenoviral-mediated gene transfer of human bone morphogenetic protein-13 does not improve rotator cuff healing in a rat model. Am J Sports Med 2011; 39:180 - 7; http://dx.doi.org/10.1177/0363546510379339; PMID: 20956264

 PubMed Web of Science ®Google Scholar

Gulotta LV, Kovacevic D, Packer JD, Deng XH, Rodeo SA. Bone marrow-derived mesenchymal stem cells transduced with scleraxis improve rotator cuff healing in a rat model. Am J Sports Med 2011; 39:1282 - 9; http://dx.doi.org/10.1177/0363546510395485; PMID: 21335341

 PubMed Web of Science ®Google Scholar

Yokoya S, Mochizuki Y, Natsu K, Omae H, Nagata Y, Ochi M. Rotator cuff regeneration using a bioabsorbable material with bone marrow-derived mesenchymal stem cells in a rabbit model. Am J Sports Med 2012; 40:1259 - 68; http://dx.doi.org/10.1177/0363546512442343; PMID: 22491821

 PubMed Web of Science ®Google Scholar

Soon MYH, Hassan A, Hui JHR, Goh JCH, Lee EH. An analysis of soft tissue allograft anterior cruciate ligament reconstruction in a rabbit model: a short-term study of the use of mesenchymal stem cells to enhance tendon osteointegration. Am J Sports Med 2007; 35:962 - 71; http://dx.doi.org/10.1177/0363546507300057; PMID: 17400750

 PubMed Web of Science ®Google Scholar

Lim JK, Hui J, Li L, Thambyah A, Goh J, Lee EH. Enhancement of tendon graft osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction. Arthroscopy 2004; 20:899 - 910; PMID: 15525922

 PubMed Web of Science ®Google Scholar

Ju YJ, Muneta T, Yoshimura H, Koga H, Sekiya I. Synovial mesenchymal stem cells accelerate early remodeling of tendon-bone healing. Cell Tissue Res 2008; 332:469 - 78; http://dx.doi.org/10.1007/s00441-008-0610-z; PMID: 18418628

 PubMed Web of Science ®Google Scholar

Shahab-Osterloh S, Witte F, Hoffmann A, Winkel A, Laggies S, Neumann B, Seiffart V, Lindenmaier W, Gruber AD, Ringe J, et al. Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions). Stem Cells 2010; 28:1590 - 601; http://dx.doi.org/10.1002/stem.487; PMID: 20882636

 PubMed Web of Science ®Google Scholar

Ouyang HW, Goh JCH, Lee EH. Use of bone marrow stromal cells for tendon graft-to-bone healing: histological and immunohistochemical studies in a rabbit model. Am J Sports Med 2004; 32:321 - 7; http://dx.doi.org/10.1177/0095399703258682; PMID: 14977654

 PubMed Web of Science ®Google Scholar

Karaoglu S, Celik C, Korkusuz P. The effects of bone marrow or periosteum on tendon-to-bone tunnel healing in a rabbit model. Knee Surg Sports Traumatol Arthrosc 2009; 17:170 - 8; http://dx.doi.org/10.1007/s00167-008-0646-3; PMID: 18941736

 PubMed Web of Science ®Google Scholar

Chen CH, Chen WJ, Shih CH, Yang CY, Liu SJ, Lin PY. Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits. Arthroscopy 2003; 19:290 - 6; http://dx.doi.org/10.1053/jars.2003.50014; PMID: 12627154

 PubMed Web of Science ®Google Scholar

Chang CH, Chen CH, Su CY, Liu HT, Yu CM. Rotator cuff repair with periosteum for enhancing tendon-bone healing: a biomechanical and histological study in rabbits. Knee Surg Sports Traumatol Arthrosc 2009; 17:1447 - 53; http://dx.doi.org/10.1007/s00167-009-0809-x; PMID: 19440695

 PubMed Web of Science ®Google Scholar

Chen CH, Chang CH, Wang KC, Su CI, Liu HT, Yu CM, Wong CB, Wang IC, Whu SW, Liu HW. Enhancement of rotator cuff tendon-bone healing with injectable periosteum progenitor cells-BMP-2 hydrogel in vivo. Knee Surg Sports Traumatol Arthrosc 2011; 19:1597 - 607; http://dx.doi.org/10.1007/s00167-010-1373-0; PMID: 21327764

 PubMed Web of Science ®Google Scholar