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Title: Human platelet rich plasma as a xenogenic platelet-rich plasma effects on critical size bone defect healing in rabbit model: Clinical, radiological and biomechanical evaluation

Authors: Z, Shafiei-Sarvestani1., A, Meimandi Parizi2., A, Oryan3., A. S. Bigham4

ID: 296195-2011

The Editor must ensure that the OJVR publishes only papers which are scientifically sound. To achieve this objective, the referees are requested to assist the Editor by making an assessment of a paper submitted for publication by:

(a) Writing a report on the reverse side of this form,
(b} Check the boxes shown below under 1. and 2. ( YES or NO) [N.B.A "NO" assessment must be
supported by specific comment in the report.
(c) Make a recommendation under 3.

The Editor-in-Chief would appreciate hearing from any referee who feels that he/she will be unable to review a manuscript within two weeks.

1. CRITERIA FOR JUDGEMENT (Mark "Yes" or "No").

Is the work scientifically sound? Y
Is the work an original contribution? No

Are the conclusions justified on the evidence presented? Y
Is the work free of major errors in fact, logic or technique? Y
Is the paper clearly and concisely written?No
Do you consider that the data provided on the care and use of animals (See Instructions to Contributors) is sufficient to establish that the animals used in the experiments were well looked after, that care was taken to avoid distress, and that there was no unethical use of animals? Animal Ethic statement needs to include POST OPERATIVE CARE STATEMENT.

2 PRESENTATION (Mark "Yes" or "No").

Does the title clearly indicate the content of the paper? NO (see suggestions)
Does the abstract convey the essence of the article? No (see changes)
Are all the tables essential? Y
Are the figures and drawings of good quality? NA
Are the illustrations necessary for an understanding of the text? Y
Is the labelling adequate? Y

3. RECOMMENDATIONS(Mark one with an X)

Not suitable for publication in the OJVR
Reassess after major changes X
Accept for publication with minor changes
Accept for publication without changes

4.REPORT: Authors report effects of human RPR on obviously artificially fractured rabbit long bone healing. There is a major ethical problem here and the authors although having provided a statement will need to provide more proof of AFTER care procedures. The work per se seems complete with both statistical measurement and photographic evidence which seem to support the findings that in this case, hPRP aided in the healing process. It would have been helpful to ascertain the purity of the hPRP in this case or at least provide a measure of its activity/content re hPRP. The Abstract has too much irrelevant information and could be summarized considerably with some thought (see suggestions). The Background is complete but the Materials and Methods are hard to follow and a more concise description of the induced wound will be required re: Ethics please). Some suggestion for this section are attached below. The results section is very confusing. I think authors should just stick to the overall findings and re-write that section bearing this is mind. There is no need for headings for each section and authors do not need to repeat results shown on Tables 1-6, just refer to them. The discussion is OK. Reconsider after major review. BR.

ABSTRACT

A lot of studies have been performed to investigate the effect of Platelet-rich plasma (PRP) upon bone defect regeneration. Platelet-rich plasma is clinically used as an autologous blood product to stimulate bone formation in vivo. The aim of the present study was to assess the effects of human PRP (xenogenic PRP) on new bone formation in a critical diaphyseal long bone defect in rabbit model. A critical size defect (10 mm) in the radial diaphysis of 12 rabbit was created and then supplied with human PRP (treatment group) or the defect left empty (control group). Platelets in the PRP were about 10.1 fold compared to normal blood. Radiographs of each forelimb was taken postoperatively on 1st day and at the 2nd, 4th, 6th and 8th weeks post injury to evaluate bone formation, union and remodeling of the defect. The operated radiuses were removed on 56th postoperative day and were evaluated for gross signs of healing. In addition, biomechanical test was conducted on the operated forearms of the rabbits. This study demonstrated that human PRP (hPRP), as a xenogenic PRP, could promote bone regeneration in critical size defects with a high regenerative capacity. The results of the present study demonstrated that hPRP could be an attractive alternative for reconstruction of the major diaphyseal defects of the long bones in animal models.

KEY WORDS: human platelet-rich plasma, radius, xenogenic PRP, bone healing, rabbit

Recommendation. Platelet-rich plasma (PRP) is used to stimulate bone formation in vivo. The effect of human PRP (xenogenic PRP) on new bone formation in diaphyseal long bone defects in rabbits is described. A critical size defect (10 mm) in the radial diaphysis was created in fully anaesthetized rabbits given human PRP (treated = 6) or not (controls = 6). Radiographs of forelimbs were taken on the 1st day and 2nd, 4th, 6th and 8th weeks post surgery to evaluate bone formation, union and remodeling. Operated radiuses were removed 56th post-surgery day and evaluated for healing and biomechanical tests were conducted on the forearm. Radiographic evidence and statistical tests suggested that human PRP (hPRP) promoted bone regeneration in the rabbit.

INTRODUCTION

Large bone defects resulting from trauma, tumors, osteitis, implant loosening or corrective osteotomies require surgical therapy, because spontaneous regeneration is limited to relatively small defects. Autogenous bone grafting is considered the gold standard for filling bone defects even today, despite significant problems arising from donor-site morbidity and limited amount of donor bone 1,2. Currently, transplantation of autografts or allografts, mineral bone substitutes 3-5and callus distraction are the most commonly used techniques for skeletal reconstruction and each of these procedures have its own significant limitations such as lack of availability or due to biological or biomechanical reasons 1,6. Therefore, osteoinductive stimulation of bone formation has received increasing interest. Both demineralized bone matrix and growth factors have been used in numerous experimental and clinical defect situations 7.

A number of growth factors are present in PRP, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and insulin like growth factor (IGF) and they have a stimulating effect on healing of the bone defects. This stimulating effect is resulted due to chemotaxis induction as well as proliferation and differentiation of osteoblasts and their precursors 7,8. An easy and more physiological way of application of growth factors to bone defects is via the use of platelet-rich plasma (PRP), a thrombocyte concentrate made up of autogenous blood 8,9.

Several previous investigations demonstrated a positive effect of PRP on wound healing 10-12. A lot of studies have been conducted to investigate the effects of PRP upon regeneration of the bone defects 9,13-19. However, the results of these studies are controversial. Marx et al. (1998) used PRP for reconstruction of the maxillofacial defects in humans and found that PRP resulted in a quicker maturation of autogenous bone transplants and higher bone density 9. Another prospective study also reported a positive effect of PRP in a similar defect situation 14. Further clinical investigations suggested an osteogenic potential of PRP but did not include control groups 15,17,18 or could not identify any positive effect 20. It should be stated that not only the clinical data are contradictory, but in vivo experimental findings are also inconsistent. In a bone defect in the iliac crest of dogs, PRP combined with demineralized bone powder enhanced bone formation around the titanium implants 21. In a rabbit skull model, however, PRP did not influence bone healing 13. In a similar study in pigs, PRP enhanced bone density temporarily when applied together with autograft but not in conjunction with a collagen scaffold containing additional osteoinductive proteins 19. Because of the controversial results, there is still need for further research regarding the possible osteogenic potency of PRP particularly with a xenogenic PRP in diaphyseal bone healing model. Therefore, the objective of this in vivo study was to investigate if the human PRP (hPRP) is effective in the reconstruction of the diaphyseal long bone defects in rabbit model. For this purpose, the human PRP were filled in a critical size defect of the rabbit radius. New bone formation was investigated after 8 weeks by clinical, radiological and biomechanical evaluations.

Recommendations re INTRODUCTION

A number of growth factors are present in PRP, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and insulin like growth factor (IGF) which have a stimulating effect on healing of the bone defects. This stimulating effect is resulted due to chemotaxis induction as well as proliferation and differentiation of osteoblasts and their precursors 7,8. An easy and more physiological way of application of growth factors to bone defects is via the use of platelet-rich plasma (PRP), a thrombocyte concentrate made up of autogenous blood 8,9.

It should be stated that not only the clinical data are contradictory, but in vivo experimental findings are also inconsistent. In a bone defect in the iliac crest of dogs, PRP combined with demineralized bone powder enhanced bone formation around the titanium implants 21. In a rabbit skull model, however, PRP did not influence bone healing 13. In a similar study in pigs, PRP enhanced bone density temporarily when applied together with autograft but not in conjunction with a collagen scaffold containing additional osteoinductive proteins 19. Because of the controversial results, there is still need for further research the osteogenic potency of PRP particularly with a xenogenic PRP in diaphyseal bone healing model. Therefore, the objective of this in vivo study was to investigate if the human PRP (hPRP) is effective in the reconstruction of the diaphyseal long bone defects in rabbit model. For this purpose, the human PRP were filled in a critical size defect of the rabbit radius. New bone formation was investigated after 8 weeks by clinical, radiological and biomechanical evaluations.

MATERIALS AND METHODS

ANIMALS AND OPERATIVE PROCEDURE

Twelve New Zealand white rabbits (12 months old, mixed sex, weighing 2.0±0.5 kg) were kept in separate cages, fed a standard diet and allowed to move freely during the study. The animals divided randomly into 2 equal groups as treated (hPRP) and control. All the animals were anesthetized by intramuscular administration of 40 mg/kg ketamine hydrochloride and 5mg/kg xylazine. In all animals the right forelimb was prepared aseptically for operation. A 5 cm skin incision was made over the forearm craniomedially and then the radius was exposed by dissecting the surrounding muscles. A 10 mm segmental defect was then created on the middle portion of each radius as a critical size bone defect. On day 4 postoperative, 1 ml hprp was injected percutaneously into the defect of bones in the treatment group while the control group was not received it. The animals were housed in compliance with our institution’s guiding principles ‘‘in the care and use of animals’’. The local Ethics Committee for animal experiments approved the design of the experiment

PRP PREPARATION

Human PRP was prepared and supplied by the Shiraz Blood bank Center. About 500 ml blood from a healthy donor was collected in 70 ml of anticoagulants (citrate-phosphate-dextrose [CPD]) and cooled to about 22 ºC. Within 24 h of extraction, the blood was separated through centrifugation into erythrocytes, buffy coat (leukocytes and thrombocytes) and plasma. From the buffy coat the leukocytes were removed through filtration, and the isolated fraction of platelets was human PRP. To obtain information on the increase in platelet concentration and the final concentration of platelets in the PRP of the obtained blood, both whole blood and prepared PRP were subjected to platelet counts. Platelet counts were performed using a hematology analyzer (Advia 120, Bayer B.V., Mijdrecht, the Netherlands). Number of platelets in whole blood was 239X109/l and in PRP was 2422X109/l.

POST OPERATIVE EVALUATION

RADIOLOGICAL EVALUATION

Radiographs of each forelimb was taken postoperatively on 1st day and at the 2nd, 4th, 6th and 8th weeks to evaluate bone formation, union and remodeling of the defect. The results were scored using a modified Lane and Sandhu scoring system 22(Table 1).

GROSS NECROPSY EVALUATION

The radial bones of rabbits (operated) were removed on 56th postoperative day; at this time the operated radius was evaluated for gross signs of healing. The examination and scoring of blinded specimens included presence of bridging bone, indicating a complete union (+3 score), presence of cartilage, soft tissue or cracks within the defect indicating a possible unstable union (+ 1 or +2 score), or complete instability at the defect site indicating no union (0 score).

BIOMECHANICAL EVALUATION

The biomechanical test was conducted on the operated bones of each rabbit. The three-point bending test was performed to determine the mechanical properties of bones. The bones were placed horizontally on two rounded supporting bars located at a distance of 30 mm, and were loaded at the midpoint of the diaphysis by lowering the third bar. The bones were loaded at a rate of 1 cm/min until fracturing occurred. Tests were performed using an universal tensile testing machine (Instron, London, UK) 23-25. Data derived from the load deformation curves were expressed as the mean ±SEM for each group.

STATISTICAL ANALYSIS

The radiological and clinical data were compared by Kruskal-Wallis, non- parametric ANOVA, when P-values were found to be less than 0.05, then pair wise group comparisons was performed by Mann-Whitney U test. The biomechanical data was compared by a student's t-test (SPSS version 17 for windows , SPSS Inc, Chicago, USA).

RESULTS

Suggestions write as follows:

RESULTS

There was a significant difference in bone formation between controls and treated on the 14th 28th and 42nd day. By day 56, there had been 100% bone formation in the animals of the hPRP group and 50-75% bone formation in those of the control group (Table 2) (Fig 1).

No animal was died during operation or until the end of the experiment and all the animals completed the study without any complications.

Radiographic findings

1. Bone formation

There was 0-25% bone formation in some rabbits in control group, however 25-50% bone formation was observed in group hPRP on 14th postoperative day. Statistical tests supported significant difference on 14 th postoperative day for bone formation.

There was significantly more (50-75%) bone formation activity in rabbits of hPRP group compared to those of the control group (0-25% bone formation) on 28th postoperative day. On 42nd postoperative day there was 75-100% bone formation in all rabbits in hPRP group while 25-75% bone formation was seen in rabbits of the control group. There was 100% bone formation in the animals of the hPRP group and 50-75% bone formation in those of the control group on 56th post operative day (Table 2) (Fig 1).

2. Bone union

There was union in rabbits of hPRP group and there was no evidence of union in the rabbits of the control group on 14th postoperative days. In addition, there was significant bone union in rabbits of hPRP group compared to those of the control ones on 28th postoperative days. There was statistically significant difference for bone union at the 42nd and 56th post operative days in the radiological signs of bone union between hPRP and control group (P<0.05) (Tables 3 and 4) (Fig. 1).

3. Remodelling

Remodeling was not found in either group on 14th, 28th and 42nd postoperative days. On 56th postoperative day remodeling was observed in rabbits of group and statistical tests revealed significant difference between the two groups, and the operated area of hPRP showed a more advanced remodeling compared to those of the control one (Table 5) (Fig 1).

MACROSCOPIC FINDINGS

The defect sites of all rabbits at necropsy contained new bone; however, the defects left blank or generally contained the least amount of new bone and were often filled with a mixture of fibrous connective tissue and cartilage. The union scores of the rabbits administered with hPRP were statistically superior to control group and their values were greater than the control animals (Table 1, p=0.001). The union score at macroscopic level correlated closely with the radiographic union score at day 56.

Biomechanical findings

There was statistical significant difference (P=0.01) between the operated bone of the control group (38.67±7.074, Mean±SEM) and that of the hPRP group (99.17±19.10, Mean±SEM) in terms of ultimate strength of the biomechanical bending test (Fig. 2).

DISCUSSION:

In this study a defect model on the radial bone was used to evaluate the bone healing with human PRP in rabbits. This model has previously been reported suitable because there is no need for internal or external fixation which influences the healing process 26. The segemental defect was created on the middle portion of the radius as long as 10 mm for inducing nonunion and to prevent spontaneous and rapid healing 27.

This study was performed to provide an explanation for the existing confusion in the literature regarding the efficacy of PRP treatment, and to give more insight into the effect of PRP on bone regeneration. To the authors’ knowledge this is one of the first studies, which presents new data on the bone regenerative properties of human PRP as a xenogenic PRP effects on bone healing in rabbit model.

The clinical and experimental data in the literature regarding the osteogenic potential of PRP are controversial. The results of the present investigation confirm a number of clinical and experimental studies demonstrating a positive influence of PRP on bone regeneration 9,14,19,28. However, in human maxillofacial defects, neither autograft nor allograft or a mineral bone substitute material enhanced bone formation when augmented with PRP 20,29,30. In a non-critical rabbit skull defect, PRP was not superior to the empty defect nor did PRP increased bone formation by autogenous bone 13.

The results of the present study indicate that hPRP stimulates a favorable reaction in the injured area of the long bones. The radiographic evaluation at 2-week post-injury showed that the bone gap was healed in the hPRP group before that of the control group and it was also already in the remodeling stage. While the defect of control animals even at the end of eight weeks post-injury were still in the healing stage. This fact was corroborated by macroscopic and biomechanical data analysis, which showed that osteogenesis in the animals of hPRP group at 56 days post-injury was stronger than those of the control group.

PRP contains several growth factors including isomers of PDGF, TGF-X 1, TGF-X 2, IGF-I, IGF-II and VEGF that all of them are promotors of bone regeneration. PDGF has been shown to be mitogenic for osteoblasts 31 and stimulates migration of the mesenchymal progenitor cells 32. It is stated that in the bone defects of the animal models, PDGF induced callus formation 16. TGF-X also has a stimulative effect on osteogenesis and inhibits bone resorption 33. In addition, it is reported that IGF-I and the angiogenic factor VEGF induced bone formation in rats 34 and in rabbits 35, respectively. In summary, these growth factors support bone regeneration primarily via chemotactic and mitogenic effects on preosteoblastic and osteoblastic cells. Due to this phenomenon, an enhanced bone formation criteria in the animals of the hPRP group compared to those of the control ones was observed. However, hPRP does not contain BMPs, the most potent osteoinductive proteins, which promote stem cell differentiation into the osteoblastic lineage and are the only growth factors known to induce ectopic bone formation 36.

Schlegel et al. (2004) and Thorwarth et al. (2005) got better results by administering higher doses of hPRP (6.5-fold compared to normal blood) than with lower platelet concentrations (4.1-fold) on bone regeneration in skull defects of minipigs 19,28. Other experimental studies found no correlation between the platelet concentration and the observed biological effects 13,21. In the present study, high platelet concentrations (10.1-fold compared to normal blood) were effective and lead to superior and faster bone formation in comparison with control group.

CONCLUSIONS

In conclusion this study demonstrated that hPRP as a xenogenic PRP could promote bone regeneration in critical size defects with a high regenerative capacity. This finding will nominate hPRP as an attractive alternative for reconstruction of the major diaphyseal defects in the long bones in animal models. Combination of functional biomaterials or autografts, precursor cells or osteoinductive growth factors with hPRP in animal models, in the future studies, may introduce more effective therapeutic regimes in regeneration and bone formation of the long bone injuries.

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3. Emami MJ, Oryan A, Saeidinasab H, et al: The effect of bone marrow graft on bone healing: A radiological and biomechanical study. Iranian Journal Medical Science 27:63-66, 2002.

4. Meimandi Parizi A, Jelodar G, Moslemi H, et al: Influence of hydroxyapatite on fracture healing in diabetic rats: biomechanical and radiographic studies. Veterinarski Arhiv 80:113-120, 2010.

5. Meimandi Parizi A, Zeidabadi nejad GR: Biomechanical and radiographical evaluation of the effects of constant direct current on the fracture healing of the radius in the rabbits. Journal Faculty of Vet Med Univ of Tehran 52:1-10, 1997.

6. Hollinger JO, Brekke J, Gruskin E, et al: Role of bone substitutes. Clin Orthop Relat Res 324:55-65, 1996.

7. Bostrom MP, Saleh KJ, Einhorn TA: Osteoinductive growth factors in preclinical fracture and long bone defects models. Orthop Clin North Am 30:647-658, 1999.

8. Weibrich G, Kleis WK, Hafner G, et al: Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg 30:97-102, 2002.

9. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:638-646, 1998.

10. McClain SA, Simon M, Jones E, et al: Mesenchymal cell activation is the rate-limiting step of granulation tissue induction. Am J Pathol 149:1257-1270, 1996.

11. Mustoe TA, Pierce GF, Morishima C, et al: Growth factorinduced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model. J Clin Invest 87:694-703, 1991.

12. Saba AA, Freedman BM, Gaffield JW, et al: Topical platelet-derived growth factor enhances wound closure in the absence of wound contraction: an experimental and clinical study. Ann Plast Surg 49:62-66, 2002.

13. Aghaloo TL, Moy PK, Freymiller EG: Investigation of platelet-rich plasma in rabbit cranial defects: a pilot study. J Oral Maxillofac Surg 60:1176-1181, 2002.

14. Anitua E: Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants 14:529-535, 1999.

15. Kassolis JD, Rosen PS, Reynolds MA: Alveolar ridge and sinus augmentation utilizing platelet-rich plasma in combination with freeze-dried bone allograft: case series. J Periodontol 71:1654-1661, 2000.

16. Nash TJ, Howlett CR, Martin C, et al: Effect of platelet-derived growth factor on tibial osteotomies in rabbits. Bone 15:203-208, 1994.

17. Robiony M, Polini F, Costa F, et al: Osteogenesis distraction and platelet-rich plasma for bone restoration of the severely atrophic mandible: preliminary results. J Oral Maxillofac Surg 60:630-635, 2002.

18. Rodriguez A, Anastassov GE, Lee H, et al: Maxillary sinus augmentation with deproteinated bovine bone and platelet rich plasma with simultaneous insertion of endosseous implants. J Oral Maxillofac Surg 61:157-163, 2003.

19. Schlegel KA, Donath K, Rupprecht S, et al: De novo bone formation using bovine collagen and platelet-rich plasma. Biomaterials 25:5387-5393, 2004.

20. Froum SJ, Wallace SS, Tarnow DP, et al: Effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts: three bilateral case reports. Int J Periodont Restor Dent 22:45-53, 2002.

21. Kim SG, Kim WK, Park JC, et al: A comparative study of osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet-rich plasma. J Oral Maxillofac Surg 60:1018-1025, 2002.

22. Lane JM, Sandhu HS: Current approach to experimental bone grafting. Orthop Clin North Am 18:213-225, 1987.

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Table 1. Modified Lane and Sandhu radiological scoring system

Bone formation

No evidence of bone formation 0

Bone formation occupying 25% of the defect 1

Bone formation occupying 50% of the defect 2

Bone formation occupying 75% of the defect 3

Bone formation occupying 100% of the defect 4

Union (proximal and distal evaluated separately)

No union

Possible union 1

Radiographic union 2

Remodeling

No evidence of remodeling 0

Remodeling of medullary canal 1

Full remodeling of cortex 2

Total point possible per category

Bone formation

Proximal union 2

Distal union 2

Remodeling 2

Maximum Score 10

Table 2. Radiographical findings for bone formation at various post-operative intervals

Med (min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-1) 1(0-2)b 0.001

28 1(0-1) 2(1-3)c 0.002

0.002

0.001

42 1(0-3) 3(1-4)d

56 2(1-3) 3(2-4)e

Significant P-values are presented in bold face.

a Kruskal-Wallis non-parametric ANOVA

bP= 0.002 (compared with control by Mann-Whitney U test)

cP= 0.007 (compared with control by Mann-Whitney U test)

dP= 0.002 (compared with control by Mann-Whitney U test)

eP= 0.001 (compared with control by Mann-Whitney U test)

Table 3. Radiographical findings for proximal union at various post-operative intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-0) 0(0-2) 0.03

28 1(0-1) 2(0-2)b 0.008

0.001

42 1(0-1) 2(1-2)c

56 1(0-2) 2(1-2)d

Significant P-values are presented in bold face

a Kruskal-Wallis non-parametric ANOVA

bP= 0.01 (compared with the control group by Mann-Whitney U test)

cP= 0.001 (compared with the control group by Mann-Whitney U test)

dP= 0.002 (compared with the control group by Mann-Whitney U test)

Table 4. Radiographical findings for distal union at various post-operative intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-1) 1(0-1)b 0.004

28 1(0-1) 2(0-2)c 0.002

0.005

0.04

42 2(0-2) 2(0-2)d

56 2(0-2) 2(0-2)

Significant P-values are presented in bold face

a Kruskal-Wallis non-parametric ANOVA

bP= 0.01 (compared with the control group by Mann-Whitney U test)

cP= 0.03 (compared with the control group by Mann-Whitney U test)

dP= 0.007 (compared with the control group by Mann-Whitney U test)

Table 5. Radiographical findings for remodeling over various post-injury intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-0) 0(0-0) 1.000

28 0(0-0) 0(0-1) 0.3

0.03

0.01

42 0(0-0) 0(0-1)

56 0(0-1) 1(0-2)b

Significant P-values are presented in bold face

a Kruskal-Wallis non-parametric ANOVA

bP= 0.01 (compared with group II by Mann-Whitney U test)

Table 6. Bone measurements at macroscopic level

Group macroscopic uniona

med(min-max)

Control (n=6) 1 (1-2)

hPRP (n=6) 3 (1-3)b

a complete union (+3 score), presence of cartilage, soft tissue or cracks within the defect indicating a possible unstable union (+ 1 or +2 score), complete instability at the defect site indicating nonunion (0 score)

b P= 0.001 (compared with the control group by Mann-Whitney U test)

Figure 1- Radiographs of forelimb, 14th postoperative day (a- control, b- hPRP), 28th postoperative day (c- control, d- hPRP), 42nd postoperative day (e-control, f- hPRP) and 56th postoperative day (g-control, h-hPRP)

Figure 2. The ultimate strength of the injured radius of the animals of hPRP group, On day 56 post-injury, is significantly greater (*) than those of the control animals (p=0.01).

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ABSTRACT

KEY WORDS: human platelet-rich plasma, radius, xenogenic PRP, bone healing, rabbit

MATERIALS AND METHODS

Animal Ethics Statement

“The animals were housed in compliance with our institution’s guiding principles ‘‘in the care and use of animals’’. The local Ethics Committee for animal experiments approved the design of the experiment” Twelve New Zealand white rabbits (12 months old, mixed sex, weighing 2.0±0.5 kg) were kept in separate cages, fed a standard diet and allowed to move freely during the study. The animals divided randomly into 2 equal groups as treated (hPRP) and control. All the animals were anesthetized by intramuscular administration of 40 mg/kg ketamine hydrochloride and 5mg/kg xylazine. No animal died during surgery or after and all the animals completed the study without any complications.

Animals and surgery

The right forelimb of all the rabbits was prepared aseptically for operation. A 5 cm skin incision was made over the forearm craniomedially and then the radius was exposed by dissecting the surrounding muscles. A 10 mm segmental defect was then created on the middle portion of each radius as a critical size bone defect. On day 4 postoperative, 1 ml hRPR was injected percutaneously into the defect of bones in the treatment group.

hPRP

Radiology

Table 1. Modified Lane and Sandhu radiological scoring system

Bone formation

No evidence of bone formation 0

Bone formation occupying 25% of the defect 1

Bone formation occupying 50% of the defect 2

Bone formation occupying 75% of the defect 3

Bone formation occupying 100% of the defect 4

Union (proximal and distal evaluated separately)

No union

Possible union 1

Radiographic union 2

Remodeling

No evidence of remodeling 0

Remodeling of medullary canal 1

Full remodeling of cortex 2

Total point possible per category

Bone formation

Proximal union 2

Distal union 2

Remodeling 2

Maximum Score 10

RESULTS

Table 2. Radiographical findings for bone formation at various post-operative intervals

Med (min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-1) 1(0-2)b 0.001

28 1(0-1) 2(1-3)c 0.002

0.002

0.001

42 1(0-3) 3(1-4)d

56 2(1-3) 3(2-4)e

Significant P-values are presented in bold face.a Kruskal-Wallis non-parametric ANOVA, (compared with control by Mann-Whitney U test) bP= 0.002, cP= 0.007, dP= 0.002, eP= 0.001

Bone union had occurred in treated rabbits by day 14th post-surgery, but not in controls. This trend continued with less union occurring in controls. (Tables 3 and 4, below) and (Fig. 1, above).

Table 3. Radiographical findings for proximal union at various post-operative intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-0) 0(0-2) 0.03

28 1(0-1) 2(0-2)b 0.008

0.001

42 1(0-1) 2(1-2)c

56 1(0-2) 2(1-2)d

Significant P-values are presented in bold face, a Kruskal-Wallis non-parametric ANOVA, bP= 0.01 cP= 0.001 dP= 0.002 (compared with the control group by Mann-Whitney U test).

Table 4. Radiographical findings for distal union at various post-operative intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-1) 1(0-1)b 0.004

28 1(0-1) 2(0-2)c 0.002

0.005

0.04

42 2(0-2) 2(0-2)d

56 2(0-2) 2(0-2)

Significant P-values are presented in bold face, a Kruskal-Wallis non-parametric ANOVA, bP= 0.01,, cP= 0.03 dP= 0.007 (compared with the control group by Mann-Whitney U test).

Remodeling was not found in either group on 14th, 28th and 42nd day. On the 56th day remodeling occurred in both groups but was more advanced in treated group (Table 5) (Fig 1).

Table 5. Radiographical findings for remodeling over various post-injury intervals

Med(min-max)

Postoperative days

Control (n=6)

hPRP (n=6)

14 0(0-0) 0(0-0) 1.000

28 0(0-0) 0(0-1) 0.3

0.03

0.01

42 0(0-0) 0(0-1)

56 0(0-1) 1(0-2)b

Significant P-values are presented in bold face, a Kruskal-Wallis non-parametric ANOVA, bP= 0.01 (compared with group II by Mann-Whitney U test)

Table 6. Bone measurements at macroscopic level

Group macroscopic uniona

med(min-max)

Control (n=6) 1 (1-2)

hPRP (n=6) 3 (1-3)b

Figure 2. The ultimate strength of the injured radius of the animals of hPRP group, On day 56

post-injury, is significantly greater (*) than those of the control animals (p=0.01).

DISCUSSION

Bone healing with human PRP in rabbits was evaluated. This model has previously been reported suitable because there is no need for internal or external fixation which influences the healing process 26. The segmental defect was created on the middle portion of the radius as long as 10 mm for inducing nonunion and to prevent spontaneous and rapid healing 27. This study was performed to provide an explanation for the existing confusion in the literature regarding the efficacy of PRP treatment, and to give more insight into the effect of PRP on bone regeneration. To the authors’ knowledge this may a first experiment, which presents new data on the bone regenerative properties of human PRP on bone healing in rabbits.