Histologic and Biomechanical Studies of Tendon-To-Bone Healing After Autologous and Allogeneic Bone Transplants

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1 RTICLE Histologic nd iomechnicl Studies of Tendon-To-one Heling After Autologous nd Allogeneic one Trnsplnts Ming-Wei Li, 1 Xin-She Zhou 2, 3 Abstrct Objectives: Compre histologic nd biomechnicl differences of tendon-to-bone heling between utologous nd llogeneic bone trnsplnts. Mterils nd Methods: Adult, helthy, New Zelnd white rbbits were used to estblish the extrrticulr tendon-to-bone heling model with the left hind limb trnsplnted with llogeneic bone nd the right hind limb trnsplnted with utologous bone. After 3, 6, nd 12 weeks fter the trnsplnt, the rbbits were killed to collect tendon-to-bone specimens, nd then the heling processes in tendonto-bone interfces were exmined. Results: All rbbits grew well fter incision without infection nd cn freely move. Histologic observtions 3 nd 6 weeks fter surgery nd biomechnicl test results 6 weeks fter surgery were sttisticlly different between the utologous nd the llogeneic trnsplnts (P <.05). After 12 weeks, histologic observtions nd biomechnicl test results showed no difference between the 2 trnsplnts (P >.05). Conclusions: Allogeneic bone trnsplnt hs reltively slower tendon-to-bone heling thn does utologous bone trnsplnt, but finlly llogeneic nd utologous bone trnsplnts hve the sme extent of tendon-to-bone heling. Key words: Autologous bone, Allogeneic bone, Tendonto-bone heling, Histology, iomechnics From the 1 Zozhung Municipl Hospitl, Zozhung, Shndong Province ; the 2 Deprtment of Orthopedics, nd the 3 Anhui Key Lbortory of Tissue Trnsplnttion, First Affilited Hospitl of engbu Medicl College, engbu, Anhui Province , PR Chin Acknowledgements: This work ws supported by reserch grnt from engbu Medicl College. Corresponding uthor: Xin-She Zhou, MD, Professor, Anhui Key Lbortory of Tissue Trnsplnttion, Deprtment of Orthopedics, First Affilited Hospitl of engbu Medicl College, engbu, Anhui Province , PR Chin Phone: E-mil: ljyz2006@hotmil.com Experimentl nd Clinicl Trnsplnttion (2013) 2: Introduction Tretments of bone defects minly include utologous bone trnsplnt, llogeneic bone trnsplnt, nd rtificil prostheses. Successful fixtion of self-tendon to grfts is pivotl for recovery of joint ctivity in bone defects close to joints. Prostheses implnttion cn be pplied to repir bone defects, but its wide ppliction hs been impeded by the difficulty of tendons to be biologiclly well fixed to the trnsplnted prosthesis. Tendons cn be well fixed to utologous trnsplnted bones, but the limittion of utologous bone grft source impedes its wide ppliction. 1 Allogeneic bone trnsplnt hs been used widely to tret bone defects becuse of its ese of ccess to llogeneic bone grfts from tissue bnks. However, the clinicl ppliction nd lbortory reserch of the fixtion of self-tendon to llogeneic trnsplnted bone hs been rrely reported. In this study, we exmined tendon-to-bone heling fter fixtion of self-tendon to llogeneic or utologous trnsplnted bone in rbbit model. Mterils nd Methods Experimentl nimls New Zelnd white rbbits were used. Rbbits were purchsed from Qinglongshn Animl reeding Rnch in Nnjing of Chin, nd they weighed 2.5 to 3.0 kg. All niml protocols were in conformity with the Guide for the Cre nd Use of Lbortory Animls published by the Ntionl Institutes of Helth 86-23, revised in Experimentl procedures Donor rbbits were killed by 15 ml of ir injected into the er vein. ones with cncellous bones were flushed with sline to remove the cnl contents, nd flushed gin with 70% ethnol, nd then Copyright şkent University 2013 Printed in Turkey. All Rights Reserved. DOI: /ect

2 Ming-Wei Li, Xin-She Zhou /Experimentl nd Clinicl Trnsplnttion (2013) 2: plced in 95% ethnol for 2 weeks. The donor bones were trnsferred in 70% ethnol, nd stored in 4 C refrigertor. Urethne ws injected into the er vein for nesthesi. Anesthetized rbbits were plced on their bcks nd fixed to the operting tble. An nterior medil incision to the proximl tibi ws mde to expose the ptellr ligment nd tibil tubercle. The ptellr ligment ws seprted into 2 prts in the middle, cut proximlly, nd the medil ptellr ligment ws mde to be free prt (Figure 1A). The tibi ws exposed in front nd bone defect ws opened bout 4 mm 5 mm deep to the bone mrrow cvity in the medil tibil tubercle (Figure 1). With tibil tuberosity s fulcrum, the free ptellr ligment t the flip ws plced in the bone defect (Figure 1C nd 1D), nd bsorbble sutures were used to fix the proximl ptellr tendon to the bone defect, nd then llogeneic or utologous bone ws used to fill the bone defect; the tendon ws embedded t the sme time. The left hind limbs of recipient rbbits were trnsplnted with llogeneic bones while the right hind limbs of the sme rbbits were trnsplnted with utologous bones (ilium). Three, 6, nd 12 weeks fter surgery, 3 rbbits were killed for Figure 1. Experimentl Procedures histologic exmintion t ech time point. 6 nd 12 weeks fter the trnsplnt, 12 rbbits were killed for biomechnicl exmintion t ech time point. Histologic observtion nd biomechnicl tests Knee joints were broken nd surrounding soft tissues of tendon-to-bone (including periosteum) were removed with the ptellr tendons spred. The ptellr ligments were cut off from the endpoint in the tibil tuberosity, nd the tluses were mputted bout 0.5 cm distnt from the bone defects. After specimens were wshed with sline, they were fixed with 10% neutrl formldehyde solution nd then subjected to hemtoxylin-eosin stining for histologic exmintion. For biomechnicl test, the tluses were mputted t the plce pproximtely 2 cm distnt from the bone defect, nd then immeditely, the mximl pull-out lod test ws performed. The specimens were fixed on the Instron-8874 computercontrolled universl testing mchine (Instron Corp, Cnton, MA, USA), nd the tendons were pulled with 10 mm/min to mesure its pull lods nd instntneous mximum tensile lod. sttisticl nlyses Sttisticl nlyses were performed with SPSS softwre (SPSS: An IM Compny, version 10.0, IM Corportion, Armonk, New York, USA). Dt were nlyzed with the pired t test. Vlues for P less thn.05 were considered sttisticlly significnt. Results C () The nterior medil ptellr ligment ws cut proximlly nd mde to be free. () The tibi ws exposed in front, nd the bone through (bout 4 mm 5 mm) to the bone mrrow cvity, which ws opened in the medil tibil tubercle to mke bone defect. (C) nd (D) With the tibil tuberosity s fulcrum, the free ptellr ligment t the flip ws plced in the bone defect, nd bsorbble sutures were used to fix the proximl ptellr tendon to the bone defect, nd the llogeneic or utologous bone ws used to fill in the bone defect, nd the tendon ws embedded t the sme time. D generl observtions All rbbits grew well fter trnsplnts, nd the incision hd no liquid lekge, no irrittion, nd no infection. Three weeks fter surgery, in the utologous trnsplnt group, bone slots nd tendons were wrpped round by lrge number of new bone tissues, nd there ws gp between the tendon nd the bone slot (Figure 2A); in the llogeneic trnsplnt group, bone slots nd tendons were wrpped round by smll mount of new bone tissues, nd there ws cler gp between the tendon nd the bone slot (Figure 2). Six weeks fter the opertion: in the utologous trnsplnt group, bone slots nd tendons were tightly connected by new bone tissues (Figure 3A); in the llogeneic trnsplnt group, bone slots nd tendons were loosely connected by new

3 166 Ming-Wei Li, Xin-She Zhou /Experimentl nd Clinicl Trnsplnttion (2013) 2: Exp Clin Trnsplnt bone tissues, nd the gps between the tendons nd the bone slots still existed in some prts (Figure 3). Twelve weeks fter the opertion: in both groups, bone slots nd tendons were tightly connected by new bone tissues (Figure 4). Histologic observtions Three weeks fter the opertion The utologous trnsplnt group: In the interfces between tendons nd bones, fibroblsts ctively proliferted with formtion of visible collgen fibers; osteoblsts ctively proliferted with lrge number of new bonelike tissues; nd chondrocytelike cells were visible (Figure 2A). The llogeneic trnsplnt Figure 2. Histologic Observtions of Tendon-to-one Heling 3 Weeks After one Trnsplnts Three weeks fter bone trnsplnts, the rbbits were killed to collect tendonto-bone specimens nd then hemtoxylin-eosin stining ws performed (mgnitude, 100). () In the interfces between tendons nd utologous trnsplnted bones, fibroblsts ctively proliferted with formtion of visible collgen fibers; osteoblsts ctively proliferted with lrge number of new bonelike tissues; chondrocytelike cells were visible. () In the interfces of tendons nd llogeneic trnsplnted bones, fibroblsts ctively proliferted with formtion of visible collgen fibers; osteoblsts ctively proliferted without obvious new bonelike tissues. Figure 3. Histologic Observtions of Tendon-to-one Heling 6 Weeks After one Trnsplnts Six weeks fter bone trnsplnts, the rbbits were killed to collect tendon-tobone specimens nd then hemtoxylin-eosin stining ws performed (mgnitude, 100). () In the interfces between tendons nd utologous trnsplnted bones, the numbers of osteoblsts nd fibroblsts decresed compred with the numbers of those 3 weeks fter surgery; lrge number of collgen fibers developed connecting closely with new bone tissues; no Shrpey s fibres were visible. () In the interfces of tendons nd llogeneic trnsplnted bones, the numbers of osteoblsts nd fibroblsts decresed compred to the numbers of those 3 weeks fter surgery; lrge number of collgen fibers developed connecting loosely with new bone tissues; no Shrpey s fibres were visible; few osteoclsts existed. Figure 4. Histologic Observtions of Tendon-to-one Heling 12 Weeks After one Trnsplnts Twelve weeks fter bone trnsplnts, the rbbits were killed to collect tendonto-bone specimens nd then HE stining ws performed (mgnitude, 400). () In the interfces between tendons nd utologous trnsplnted bones, few osteoblsts nd fibroblsts were seen; lrge number of collgen fibers existed fusing with bone tissues; Shrpey s fibres were visible. () In the interfces between tendons nd llogeneic trnsplnted bones, few osteoblsts nd fibroblsts were seen; lrge number of collgen fibers existed fusing with bone tissues; Shrpey s fibres were visible. group: In the interfces of tendons nd bones, fibroblsts ctively proliferted with the formtion of visible collgen fibers; osteoblsts ctively proliferted without obvious new bonelike tissues (Figure 2). Six weeks fter the opertion The utologous trnsplnt group: In the interfces between tendons nd bones, the numbers of osteoblsts nd fibroblsts decresed compred to the numbers of those 3 weeks fter surgery; lrge number of collgen fibers developed connecting closely with new bone tissues; no Shrpey s fibres were visible (Figure 3A). The llogeneic trnsplnt group: In the interfces between tendons nd bones, the numbers of osteoblsts nd fibroblsts decresed compred with the numbers of those 3 weeks fter the opertion; lrge number of collgen fibers developed connecting loosely with new bone tissues; no Shrpey s fibres were visible; few of osteoclsts existed (Figure 3). Twelve weeks fter surgery The utologous trnsplnt group: In the interfces between tendons nd bones, few osteoblsts nd fibroblsts were seen; lrge number of collgen fibers existed, fusing with bone tissues; Shrpey s fibres were visible (Figure 4A). The llogeneic trnsplnt group: In the interfces between tendons nd bones, few osteoblsts nd fibroblsts were seen; lrge number of collgen fibers existed fusing with bone tissues; Shrpey s fibres were visible (Figure 4).

4 Ming-Wei Li, Xin-She Zhou /Experimentl nd Clinicl Trnsplnttion (2013) 2: iomechnicl test (mximl pull-out lod) Six weeks fter surgery, the mximl pull-out lod in the utologous trnsplnt group ws significntly greter thn in the llogeneic trnsplnt group (Tble 1; P <.01). Twelve weeks fter the opertion, there ws no difference in the mximl pull-out lod between 2 groups (Tble 1; P >.05). The results suggest tht in the erly stge, tendon-to-bone heling in the utologous trnsplnt group is superior to heling in the llogeneic trnsplnt group, but in the lter stge, there is no difference in tendon-to-bone heling between the 2 groups. Tble 1. Mximl Pull-Out Lod Test t Different Times Test Number of Mximl Pull-Out Lod (N) Pired t Test Time Cses (n) Allogeneic trnsplnt Autologous trnsplnt P vlue 6 weeks ± ± < weeks ± ± >.05 Six weeks nd 12 weeks fter bone trnsplnts, rbbits were killed to collect tendon-to-bone specimens. knee joints were broken nd surrounding soft tissues of tendon-to-bone (including periosteum) were removed with the ptellr tendons spred. The ptellr ligments were cut off from the endpoint of the ptellr ligments in the tibil tuberosity, nd the tluses were mputted t pproximtely 2 cm from the bone defect. Specimens were fixed on the Instron-8874 computer-controlled universl testing mchine, nd then the tendons were pulled to mesure their pull lods nd instnt mximum tensile lod. Discussion one heling processes fter llogeneic nd utologous bone trnsplnts re bsiclly similr, except tht llogeneic bone heling is reltively slower probbly becuse there re no vible cells in the trnsplnted llogeneic bone fter freezing process nd thus, bone heling relies entirely on the host tissue invding nd growing into the trnsplnted bone, known s crwling substitution. 1,2 The current study exmined tendon-to-bone heling fter utologous nd llogeneic bone trnsplnts, nd showed tht llogeneic nd utologous tendon-to-bone heling processes were bsiclly similr, but llogeneic tendon-to-bone heling ws slower. Leung nd collegues found tht in the utologous tendon-bone interfce, extensive scr tissues were formed to overbridge the heling interfce nd remodel with heling over time. 3 In the current study, we found the similr phenomenon in llogeneic bone-tendon heling: the tendon nd the llogeneic bone were connected with collgen tissues, nd over time, collgen tissues were fused with the bone nd the tendon, nd then remodeled to develop norml ttchment site. Tendon enthuses cn be clssified s fibrocrtilginous or fibrous ccording to tissues present t the skeletl ttchment site. 4 Fibrocrtilginous entheses hve 4-lyer trnsitionl structure: fibrous connective tissue, fibrocrtilge, clcified fibrocrtilge, nd bone tissue. 5 A cler, continuous tide line develops between the fibrocrtilge nd the clcified fibrocrtilge. Fibrous enthuses re fibrous connective tissues between the fibrous tendon nd bone. In the present study, 3 weeks fter the opertion, fibroblst prolifertion ctivity nd the formtion of collgen fibers were observed between the tendon-bone interfce. Six weeks fter the opertion, lrge number of collgen fibers developed connecting loosely with new bone tissues. Twelve weeks fter the opertion, lrge number of collgen fibers existed, fusing with bone tissues, nd Shrpey s fibres were visible. So, fibrous enthuses were observed in our study. one defects cused by trum re common problemtic conditions. Currently, tretments for bone defects minly include utologous bone trnsplnt, llogeneic bone trnsplnt, nd rtificil prostheses. Artificil prostheses implnttion cn be pplied to repir bone defects, but it is difficult for tendons to be biologiclly fixed well to the trnsplnted prosthesis. Tendons cn be fixed well to utologous trnsplnted bones, but the limittion of utologous bone grft source impedes its wide ppliction. Allogeneic bone trnsplnt hs been used widely to tret bone defects becuse of ese of ccess to llogeneic bone grfts from tissue bnks. However, little is known bout the clinicl ppliction nd preclinicl reserch of the fixtion of self-tendon to llogeneic trnsplnted bone in bone defects close to joints. Our study showed tht llogeneic bone trnsplnt hd reltively slower tendon-to-bone heling thn did utologous bone trnsplnt in rbbit model, but ultimtely llogeneic nd utologous bone trnsplnts hd the sme level of tendon-to-bone heling, s demonstrted by histologic nd biomechnicl tests. Our study suggests tht it my be fesible to pply llogeneic bone trnsplnt to treting bone defects close to joints nd keeping joint ctivity. References 1. Perrit M, Chvrier C. Implnt bone reconstruction using llogenic bone grfts. Implnt. 2000;6(3):

5 168 Ming-Wei Li, Xin-She Zhou /Experimentl nd Clinicl Trnsplnttion (2013) 2: Exp Clin Trnsplnt 2. Schwrz N, Schlg G, Thurnher M, Eschberger J, Dinges HP, Redl H. Fresh utogeneic, frozen llogeneic, nd declcified llogeneic bone grfts in dogs. J one Joint Surg r. 1991;73(5): Leung KS, Qin L, Fu LK, Chn CW. A comprtive study of bone to bone repir nd bone to tendon heling in ptell-ptellr tendon complex in rbbits. Clin iomech (ristol, Avon). 2002;17(8): enjmin M, Kumi T, Milz S, oszczyk M, oszczyk AA, Rlphs JR. The skeletl ttchment of tendons--tendon entheses. Comp iochem Physiol A Mol Integr Physiol. 2002;133(4): Petersen W, Lprell H. Insertion of utologous tendon grfts to the bone: histologicl nd immunohistochemicl study of hmstring nd ptellr tendon grfts. Knee Surg Sports Trumtol Arthrosc. 2000;8(1):26-31.