Effect of alendronate and platelet-rich plasma in tendon-bones integration in sheep anterior cruciate ligament surgery

Context : Anterior cruciate ligament (ACL) reconstruction often faces challenges due to poor integration of the tendon with bone. Aims : To evaluate the potential benefits of alendronate and platelet-rich plasma (PRP) in enhancing tendon-to-bone osteointegration in ACL surgery. Methods : This was a post-test only control group experimental study with Ovis aries Linnaeus sheep as experimental animals. The sample was divided into four groups: the control group ACL reconstruction with calcaneal tendons given NaCl, the group given PRP, alendronate, and PRP and alendronate. Bone healing biomarkers (NF- B, TNF-α, MMP -9, TGF-β1, and COL1A1) were examined through immunohistochemical analysis and histological studies to assess osteoblast counts and inflammatory tissue. Results : There was a statistically significant (p<0.05) increase in MMP-9 and osteoblast count after alendronate and PRP administration. Administration of alendronate and PRP also increased other variables, namely TNF-α , COL1A1, and the level of inflammation, although not statistically significant (p>0.05). The intervention did not affect NF- B and TGF-β 1 (p> 0.05). Conclusions : These results show that the administration of alendronate and PRP improves the healing of tendon-calcaneal ACL reconstruction surgery in sheep.


INTRODUCTION
The anterior cruciate ligament (ACL) is a strong fibrous ligament that is minimally vascularized and located inside the knee joint, so ACL reconstruction using tendon/ligament donors is the main option for maintaining knee joint stability (Gans et al., 2018).The incidence of ACL ligament rupture itself varies.In the United States alone, 80,000 to 250,000 cases per year can be seen, with up to 50,000 cases per year of reconstructive surgery performed (Zbrojkiewicz et al., 2018).The procedure of ACL reconstruction is a highly efficacious surgical intervention, yielding a remarkable 75 to 90% of patients reporting positive outcomes categorized as good or excellent.However, it is important to note that a considerable proportion of patients (10 to 15%) may necessitate a revision procedure (Samitier et al., 2015).One of the etiologies of graft failure that continues to occur is a failure of osteointegration of the tendon to the bone (Gans et al., 2018).
So far, the treatment for ACL rupture is ACL reconstruction alone without bioengineering from platelet-rich plasma (PRP) or injection of other drugs.It is necessary to be bioengineered to prevent the failure of ACL reconstruction, so several studies have been carried out, including using alendronate (ALN) and PRP.Alendronate substantially enhances bone mass and tissue density within the bone canal.Moreover, alendronate also augments peri tunnel bone mass and density along the tunnel, resulting in superior integration of the graft tunnel in the 6th week.Alendronate diminishes peri-tunnel bone resorption, enhances tissue mineralization within the tunnel as well as histologically and biomechanically, and expedites the healing of graft tunnel, which is presumably attributable to the reduced expression of MMP-9 and CD 68positive cells (Lui et al., 2013).
PRP is a highly concentrated autologous suspension of platelets in plasma following centrifugation.This exceptional blood product is packed with an impressive selection of proteins and growth factors, which are crucial in managing the growth and maturation of a diverse range of tissues.The utilization of PRP has been shown to greatly accelerate the process of wound healing by boosting the proliferation of cells, formation of matrices, and osteoid production.It also promotes the healing of connective tissue, angiogenesis, and collagen synthesis (Gobbi and Vitale, 2012).
The balance between bone resorption and formation is critical for maintaining bone mass.The reduction in bone mass is brought about by inflammatory conditions that promote bone resorption while impeding bone formation.It has been reported that NF-B inhibitors possess anti-inflammatory properties, while also inhibiting osteoclast formation and promoting bone formation.Tumor necrosis factor alpha (TNF-α) has been found to cause the degradation of bones by stimulating osteoclast activity while hindering the differentiation of osteoblasts and inducing apoptosis.Therefore, biologic drugs targeting TNF-α are effective for treating bone damage (Zhao, 2017).TGF-β is the primary endogenous growth factor in bone healing that stimulates cellular activity, growth, differentiation, and extracellular matrix production.The TGF-β released by the platelets in the affected region after the formation of the first blood clot leads to the proliferation of periosteal cells (Boakye et al., 2015).Osteoblasts, which are in charge of bone formation, develop from precursors found in the periosteum's deeper layers and the bone marrow.They then produce an osteoid matrix, primarily type I collagen (Zhao, 2017).
Based on the background of the problems present, the study was designed to investigate the impact of alendronate and PRP on calcaneal tendon-based ACL surgery in experimental animals, specifically at the molecular level.

Experimental animals
This study has procured ethical clearance from the Research Ethics Commission of the Faculty of Veterinary Medicine, Gadjah Mada University, bearing the identifier Number 0008/EC-FKH/ Ex./2021.
The experimental study utilized Ovis aries Linnaeus sheep as experimental animals in a post-test only control group design.The study was scheduled from March 2021 to December 2022 at Lembaga Penelitian dan Pengujian Terpadu (LPPT) Universitas Gajah Mada in Yogyakarta, Indonesia.The research was conducted in collaboration with the Department of Anatomical Pathology, Dr. Moewardi Hospital, in Surakarta, Indonesia.The research subjects were male sheep, aged about two years and weighing 20 kg, obtained from the Universitas Gajah Mada.Adaptation was carried out for research subjects with appropriate cage care so they continued moving freely and did not experience stress.All research subjects were given a standard diet during the trial period.ACL reconstruction was carried out afterward.The number of samples taken was 28, divided into 4 groups.The first/control group (K0) was given only NaCl, the second group (K1) was given only alendronate, the third group (K2) was given PRP only, and the fourth group (K3) was given PRP and alendronate.After 12 weeks, the subjects were sacrificed, and the histological/chemical examination was conducted afterward.

Platelet-rich plasma
Each 10 mL of blood taken was given 1 mL of sodium citrate and then spun with 900 relative centrifugal force (RCF) for 5 minutes to form a platelet-poor plasma (PPP) layer, buffy coat, and red blood cells.The PPP layer and the buffy coat were separated with a second spin with a speed of 1500 RCF to form two layers of PPP and platelet pellets.The PRP was obtained with the homogenization of the two layers (Machado et al., 2019).

Alendronate administration
Alendronate was administered subcutaneously, with a moderate dose of 60 µg/kg, once a week for six weeks (Lui et al., 2013).

ACL reconstruction
The ACL reconstructive surgery procedure was performed surgically in a veterinary operating room.The sheep were anesthetized by the veterinarian, and then the hair around the operating area was shaved and cleaned with betadine and 70% alcohol.A sterile drape was attached to the operating area, and the knee joint was opened through a medial parapatellar incision.The semitendinosus tendon from the same side was isolated as a graft; its length and diameter were measured, and the graft was compacted with a holding suture, then the diameter of the graft was measured again.The patella was dislocated, and the prepatellar fat pad was cleaned.The original ACL was identified and cut, and the length and diameter were measured.Then, a femoral tunnel was created from the ACL footprint at a 55-degree angle to the articular surface using a K wire.A tibial tunnel was created at a 55-degree angle to the articular surface using K wire.The ends of the graft were wrapped with a collagen scaffold, and then the graft was immersed in PRP.The tendon graft was pulled into the tunnel, and the femoral and tibial ends were fixed using a bio-degradable screw with a diameter of 6-7 mm with maximum tension at 30 degrees of flexion.PRP was injected around the graft in the tunnel.The synovial and joint capsule were closed.The patellar was relocated and sutured, and then the wound was covered with a gauze dressing (Meller et al., 2008).

Evaluation of immunohistochemistry
The examination of outcomes from immunohistochemistry was executed via the utilization of an Olympus CX22 Microscope.This was carried out by a trained professional in the field of Anatomical Pa-thology, specifically an Anatomical Pathologist, within the Department of Anatomical Pathology, Sebelas Maret University.All 28 samples were observed for accumulated total expression of NF-B, TNF-α, MMP-9, TGF-β1, and COL1A1.Stained cells that exhibit immunoreactivity would be enumerated for their complete aggregation in the full field of vision, employing a magnification of 400×.Subsequently, the overall percentage would be evaluated to be employed as the percentage of NF-B, TNF-α, MMP-9, TGF-β1, and COL1A1 protein expression.Meanwhile, the number of osteoblasts and the percentage of inflammation were examined for histological inspection.

Statistical analysis
In this study, the data obtained was statistically analyzed using the Statistical Product and Service Solutions (SPSS) program for Microsoft Windows release 25.0.The number of research samples was less than 50 samples, so the Shapiro-Wilk distribution test was used to determine the distribution of expression data for all immunohistochemical variables and histological data.The statistical test used was the one-way ANOVA test to compare 4 unpaired sample groups with normal distribution.If the data was not normally distributed, it was first transformed using the root transformation so that it was normally distributed.If, after the transformation, it was still not normally distributed, the Kruskal-Wallis test was used (Dahlan, 2011).

RESULTS
Post-test histological tissue collection was carried out after the sample received ACL reconstruction and different interventions for 12 weeks at the Animal Laboratory of the Faculty of Veterinary Medicine UGM.The sample tissue was then brought to the Laboratory of Anatomical Pathology, Faculty of Medicine, Sebelas Maret University, where immunohistochemistry was used to check for the variables under investigation.All research results are presented in Table 1.All histological analysis results were provided in the attachment section after the references (see Supplementary data).
The results of immunohistochemical examination of NF-B, TNF-α, MMP-9, TGF-β1, and COL1A1 are expressed in percentage form of stained variables compared to the entire tissue.The mean NF-B percentage in the control group (K0) was 32.14 ± 25.31.While the average number of NF-B in the sample group treated with alendronate (K1), PRP (K2), and PRP with alendronate (K3) respectively were 22.07 ± 19.51, 31.79 ± 14.05, and 27.50 ± 23.80%.The mean TNF-α percentage in the control group (K0) was 31.14According to a one-way ANOVA analytical test, the NF-B and COL1A1 groups did not differ significantly.This demonstrated that these two variables did not significantly differ between groups.Further study of Tukey's post hoc test was not conducted because there were no apparent differences between the groups.
According to the results of the Kruskall-Wallis analysis test, the MMP-9 variable demonstrated that there were significant differences between the groups.So, a follow-up study of the Mann-Whitney post hoc test was performed to determine which group differences were significant.Based on the outcomes of the post hoc analysis from Table 2, notable distinctions were observed among the MMP-9 variables in the K0 vs. K2, K0 vs. K3, K1 vs. K2, and K1 vs. K3 groups.However, the K0 vs. K1 and K2 vs. K3 groups did not exhibit any significant difference.These findings suggest that the PRP-administered group invariably exhibited a noteworthy difference when compared to the non-PRP group.Nevertheless, the PRP and alendronate group did not depict any significant difference compared to the PRP group, nor did the control group exhibit any significant difference compared to the alendronate group.
The results of histological and histochemical examination were carried out by evaluating the number of osteoblasts and assessing the level of inflammation.The variable data are then displayed as mean results according to Table 3.
Based on the comparative test results that have been conducted, a significant difference (p<0.05) in the number of osteoblast cells between groups was observed (Table 4).By contrast, in terms of inflammation level, no significant difference was detected between the groups (p>0.05).This implies that in this study, neither the control group nor the groups administered with alendronate and PRP exhibited any differences in the level of inflammatory tissue.While the intervention demonstrated a significant contrast in the quantity of osteoblast cells, a post hoc followup analysis was performed to determine which differences among the groups were significant.
Based on the post hoc analysis results, there was a significant difference between the number of osteoblast cells in the K0 vs. K3 and K1 vs. K3 groups.Meanwhile, between groups K0 vs. K1, K0 vs. K2, K1 vs. K2, and K2 vs. K3 did not show significant differences.These results indicated that the combination of alendronate and PRP provided a significant difference in the number of osteoblast cells when compared to the control group (K0) or the PRP treatment group alone (K2).

DISCUSSION
According to the findings of this investigation, neither PRP nor alendronate nor their combination demonstrated a significant variance in average rates of NF-B across groups.This outcome deviates from prior research.Xin et al. (2020) discovered a reduction (p<0.01) in protein expression associated with the NF-B signaling pathway in the ALN and PRP group in comparison to the OA group.Moreover, the ALN + PRP group exhibited a greater protein expression decrease than the ALN or PRP group alone (p<0.01).This phenomenon can be attributed to ALN or PRP treatment enhancing chondrocyte viability.Notably, the ALN + PRP group displayed a higher level of chondrocyte viability than the ALN and PRP groups.Other investigations have demonstrated a positive correlation between elevated leukocyte concentrations and high interleukin-1 beta (IL-1β) levels and TNF-α in leukocyte-concentrated PRP.This rise in TNF-α is consistent with the present research outcomes, which revealed an increase in TNF-α in the PRP group, although this increase was not statistically significant.The upsurge of IL-1β and TNF-α levels plays a crucial role in the inflammatory response, leading to an escalation in NF-B (Yin et al., 2017).
This study also showed that giving PRP or PRP with alendronate increased MMP-9 statistically significantly.PRP itself is known to have high concentrations of cytokines such as TNF-⍺ and IL-1β, which positively correlates with MMP-9 concentrations.The primary function of platelet-rich plasma lies in its ability to optimize the growth factors contained within platelet ⍺-granules, aiming to foster an anabolic environment at the injury site (Sundman et al., 2011).
This study has yielded the finding that the administration of alendronate or alendronate in conjunction with PRP resulted in a reduction of TGF-β1, albeit lacking statistical significance.It has been demonstrated that the inhibition of TGF-β1 signaling can lead to an increase in bone mass and an enhancement of bone quality (Jia et al., 2013).In this study, PRP did not induce any significant alteration in TGF-β1 expression.However, a different study reported an elevation in TGF-β1 expression 12 weeks following surgery with PRP administration.TGF-β1 can stimulate mesenchymal stem cells and chondrocytes, thereby facilitating chondrogenic proliferation.Furthermore, it impedes the catabolic activity of IL-1, which promotes the degradation of the cartilage extracellular matrix (Boakye et al., 2015).
This study also showed that giving PRP increased COL1A1, although not statistically significant, this result is in line with previous research, which found PRP increased COL1A1, which was statistically significant.The increase in COL1A1 by PRP likely originates from platelet ⍺-granules.Furthermore, it is widely acknowledged that PRP possesses cartilage oligomeric matrix protein (COMP), a non-collagen pentameric glycoprotein that plays a significant role in the construction of the collagen matrix through its binding to collagen types I and II, subsequently promoting collagen fibrillogenesis (Ávila et al., 2016;Schnabel et al., 2007).
Based on the findings of the present investigation, notable variations were detected in the mean count of osteoblast cells among the groups, as inferred from the One-Way ANOVA comparative test analysis.The administration of alendronate and PRP yielded a noteworthy disparity in the average count of osteoblast cells compared to the control group (K0) and the alendronate group (K1).Nevertheless, no significant difference was observed between the PRP (K2) and alendronate with PRP (K3) groups.These results demonstrate that PRP has a significant impact on the augmentation of osteoblast cells.These conclusions are consistent with Kinoshita et al. (2020) research, which established that the administration of PRP led to a ten-fold surge in the number of osteoblasts in comparison to non-administration.This phenomenon is attributable to the elevated platelet-derived growth factor (PDGF) level present in PRP (Lee et al., 2013).The elevated content of PDGF has been observed to amplify the proliferation rate of osteoblasts through the PDGFR-mediated ERK signaling pathway (Kinoshita et al., 2020).The induction of osteoblast differentiation is achieved by PDGF through the augmentation of collagen synthesis, bone cell proliferation, and tissue repair (Shah et al., 2014).Furthermore, the aforementioned findings are in concurrence with a systematic analysis conducted by Gentile and Garcovich (2020), wherein they ascertained that PRP induced a considerable elevation in osteoblast proliferation.The research conducted by Arumnada (2021) suggests that using PRP after a 60-day interval significantly enhanced the quantity of histologically detected osteoblasts, which aligns with the results obtained in this investigation.
Based on histological studies, there was no difference in the level of inflammation in the control group (K0), the alendronate group (K1), the PRP group (K2), and the PRP and alendronate group (K3).The results obtained did not exhibit a significant difference in the inflammatory tissue in the histological studies despite indicating an increase in the intervention groups.The absence of significant differences is likely attributable to the extensive duration between the intervention and the evaluation, which allowed for the resolution of the inflammatory process.In studies concerning alendronate and its impact on inflamed tissue, administering alendronate did not elicit any substantial differences in the response of inflammatory cells and tissues observed from a histological perspective compared to the control group on days 30 and 60.These findings are analogous to those of the present study (Cengiz et al., 2005).
The findings of this research proved that the combination of PRP and alendronate significantly increases the total count of osteoblast cells, which improves tendon-to-bone healing after six weeks.These findings showed different results from a systematic review from Figueroa et al. (2015) that found there was no evidence regarding the improvement of tunnel healing with the intervention of PRP only (Figueroa et al., 2015).Nevertheless, a recent meta-analysis showed that the administration of PRP improved the clinical outcomes of ACL reconstruction assessed through VAS, IKDC, and Lysholm and Tegner scores (Zhu et al., 2022) even though further research is still needed to reduce inconsistencies in existing findings.
This study is subject to a significant limitation, namely the small size of the specimens utilized in the investigation.The researcher readily acknowledges that a more sizable sample would yield more robust and meaningful findings.Therefore, it is highly recommended that future research should consider using data obtained from a larger sample size.Despite the limitations acknowledged, the author remains confident that our research provides valuable insights into the integration of tendons and bones in the anterior cruciate ligament of sheep.

CONCLUSION
The administration of alendronate and PRP resulted in a significant increase in MMP-9 and osteoblast count, as well as TNF-α, COL1A1, and inflammation levels, except for the latter, which was not statistically significant.Additionally, the administration of alendronate and PRP exhibited a reduction in NF-B and TGF-β1.These findings demonstrate that the administration of alendronate and PRP enhances the tunnel healing process of ACL reconstruction surgery utilizing tendon-calcaneal in sheep.However, further research is required before direct application in humans can be considered.

Table 1 .
The mean percentage of the results of the immunohistochemical variable.

Table 2 .
Results of MMP-9 analysis with post hoc test Mann-Whitney.

Table 3 .
The mean result of histological variable.

Table 4 .
The analysis results of the number of osteoblast cells by post hoc test LSD.