Analysis of the shear strength of a hydroxyapatite (HA)-coated commercially pure titanium (cp-Ti) implant: A in vivo study in pigs

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Analysis of the shear strength of a hydroxyapatite (HA)-coated commercially pure titanium (cp-Ti) implant: A in vivo study in pigs

https://doi.org/10.21203/rs.3.rs-5224504/v1

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We assess the shear strength of a commercially pure titanium (cp-Ti) implant coated with hydroxyapatite (HA) in the intramedullary canal of miniature pigs' femur. The research involved the utilization of cylinders coated with cpTi-HA, which had an average diameter of 9 mm and a length of 10 mm. The interfacial strength between the bone and the implant reached an ultimate value of 11.32 MPa. The procedure of implantation was performed on three miniature pigs, and the process of osteointegration was assessed using three-dimensional images and histomorphology. In order to examine any detachment, scanning electron microscopy (SEM) was utilized to inspect the complete interface between the implant and the bone. Our discoveries revealed a failure mode that resembled the removal of the femoral stem in a hip revision procedure at the bone-to-bone interface.

Animal model

Shear strength

Commercially pure titanium

Implant

Nowadays, the utilization of commercially pure titanium (cp-Ti) with a hydroxyapatite (HA) coating has become popular in hip arthroplasty surgery, specifically in the femoral stem [1–4]. It has been demonstrated that the application of a HA coating enhances the attachment strength, while the use of commercially pure titanium (cp-Ti) exhibits favorable bonding strength and biocompatibility compared to titanium alloy materials.[1, 2, 5–11]

However, the current approach to determine shear strength requires the workpiece to undergo complete osteointegration before testing can commence. Unfortunately, this method cannot be carried out on human subjects and is thus limited to animal experimentation.

Furthermore, upon examining research conducted on animals, it is evident that the shear strength of titanium with HA coating and the resulting experimental outcomes and values display significant variations. This can be attributed to various factors, including the size of the animal, the dimensions of the implant, the location of implantation, and the material properties.

The examination of the shear strength in coatings composed of Titanium-Hydroxyapatite (Ti-HA) involves the implementation of various experiments on animals of varying sizes.

For instance, in the case of small animals like rabbits, K.A. HING et al presented findings that revealed an interfacial shear strength of 7.3 MPa in the femur of rabbits measuring D4.5mm*6.55mm at the distal femur [12]. Additionally, Stewart M reported a shear strength of 5.2 MPa [13].

Furthermore, Matthew Stewart et al examined the bond strength of HA-coated Ti-6A1-4V in the intramedullary canal (5.2 MPa); however, it is important to note that the rabbit used in the study was a small animal and the titanium rod was extremely thin (2 mm in length and 4 mm in diameter). Consequently, the interfacial shear strength yielded a notably low value.

Moving on to intermediate animals such as dogs, the interfacial shear strength was evaluated using dogs weighing between 20 and 25 kg, which displayed an interfacial shear strength of 3.4 MPa[14].

Meanwhile, they report a low value of shear strength. Joo et al report a bond strength at the bone interface. They found that the shear strength is 3 MPa and was not correlate with bone contact length and they didn’t performed in femoral canal [14]. B. C. Wang and K. Hayashi also reported the shear strength of plasma-sprayed hydroxyapatite coating implant is 13.97 MPa and 9.7 MPa respectively. Even though the shear strength might be higher than rabbits, but they performed in a transcortical site, not a femoral intramedullary canal[15, 16].

Dog V. I. Kalita et al. report a shear strength of porous titanium with HA coating, 16 weeks of implantation are 4.25–4.81 MPa, diameter 2mm femur distal femur, The limitation is that the workpiece size is small and it wasn't inserted into the intramedullary of the proximal femur, as would be done in human surgical procedures.[17]

In the case of larger animals such as sheep and pigs, William Robert has stated that the use of a HA coating serves as an osteoconductive surface for bone growth and enhances the strength of the cortical bone-implant interface, measuring at 18.9 MPa in the cortical site. Despite this increase in shear strength that is influenced by animal size, the research conducted only utilized small-sized workpieces and did not provide data on shear strength in cancellous bone.[18]

The research question and animal safety should take into account the impact factor of animal selection and implant design. When exploring the interaction between bone and implant, factors such as shear strength, osteointegration, and the size of the large animal are crucial due to their similarity to human bone in terms of anatomy, morphology, and the healing and remodeling process. However, it is important to note that there is currently no animal study that replicates material testing in human implants. Hence, this study employed a large animal model, a larger diameter implant, and the use of HA coating inserted into the intramedullary canal of the proximal femoral bone.

The objective of our study was to assess the shear strength in the femoral bone of a large animal, aiming for a close resemblance to that of humans (specifically the distal stem), while also utilizing an implant size similar to that used in hip surgery.

2.1 implant materials

In this investigation, all samples were fabricated using cpTi-HA-coating, which refers to a Ti-6Al-4V forged alloy in accordance with the DIN ISO 5832/3 standard.

Cementless stems, which are utilized in the context of Hip arthroplasty[4], can be obtained with either the implaFix® cpTi-coating or the implaFix® HA-coating. These particular samples enjoy extensive usage in the aforementioned field. A machine was employed to precisely sever the implant rods in a perpendicular manner. Each individual specimen was provided in the form of cylinders with an average diameter of 9 mm and a length of 10 mm (refer to Figure.1). The process of creating porous structures through the Titanium Plasma Spray (TPS) technique reduces the fatigue resistance to an acceptable level. The HA coating possesses a layer thickness of approximately 60 µm, a roughness of 40 µm, and an adhesive strength exceeding 15 MPa.

2.2 Implantation procedure

This investigation was conducted on three miniature specific-pathogen-free pigs (Sus scrofa domesticus), 30 months old with an approximate average weight of 30kg. We obtained approval for the study from the local ethics committee and performed the surgical component of the project at the Chulalongkorn University Laboratory Animal Center (CULAC) in Bangkok, Thailand, under IRB number 2173022. In accordance with laboratory animal care policies, the animals and our experimental study were treated in accordance with the guidelines of the CULAC-ACUP and ARRIVE guidelines 2.0. The animals were individually housed in cages within a controlled animal room, maintained at a temperature of 22 ± 2°C and a relative humidity of 50 ± 20%, with standard fluorescent lighting and ventilation occurring 15–20 times per hour. Additionally, a 12-hour light-dark cycle was maintained. The animals had unrestricted access to both diet and water. A commercial pig feed was provided twice daily at a rate of 1–2% of the animal's body weight in pellet form.

Anesthetic induction was conducted using injectable anesthesia, consisting of 5 mg/kg tiletamine-zolazepam (Zoletil, Virbac, Thailand), 2.5 mg/kg ketamine (Alfasan Nederland BV, Netherlands) and 12.5 mg/kg xylazine (X-Lazine, L.B.S. Laboratory Ltd, Thailand). Following induction, the auricular vein was catheterized for the administration of normal saline solution. All pigs underwent endotracheal intubation, receiving 100% oxygen to maintain optimal oxygen levels. Anesthesia was maintained throughout the procedure using an appropriate concentration of isoflurane for the surgery. Cefazolin (25 mg/kg) was administered intravenously as prophylaxis antibiotic and repeated every 90 minutes. Craniodorsal approach through hip joint was performed by craniolateral incision as previously described (Johnson). Skin was incised caudally over the trochanter of the femur. Subcutaneous tissues and fascia lata were reflected. The biceps femoris muscle was retracted caudally, allowing for identification of the sciatic nerve. The superficial gluteal muscle was then be retracted craniodorsally, facilitating reaming of the femoral canal from a superior direction. The diameter of the drill was smaller than that of the implant by 2 mm. Intramedullary reaming was performed with sterile saline irrigation to insert the implant using a press-fit technique. The placement of the femoral implant was guided by x-ray, with three implants being inserted into each femur. (Figure.2) Subcutaneous will be closed with monosyn 0 or 2/0 and skin will be closed with 2/0 nylon sutures. After surgery, all pigs received cefazolin 25 mg/kg intramuscularly injection daily for 7 days. Carprofen (Rimadyl®, Laboratorios Pfizer LTDA, Guarulhos, Sao Paulo, Brazil) at dose 3 mg/kg will be used as analgesic and subcutaneously injected daily after operation for 5 days. Wound dressing will be changed, and betadine will be applied daily. Stitches will be removed at 10 days after surgery.

Clinical signs and pain were carefully monitored. After a period of three months, all miniature pigs were humanely euthanized for sample collection. Premedication and anesthesia were conducted following the same protocol as used for surgery, ensuring that each animal reached a deep plane of anesthesia. Throughout the process, animals were closely monitored to prevent any pain or distress. Once fully anesthetized, 75–150 mg/kg of potassium chloride (KCl) was administered intravenously. Vital signs, including heart rate and respiratory rate, were monitored to confirm the absence of cardiovascular function, ensuring complete euthanasia. The femoral bones were dissected and cut under fluoroscopy. (Figure.3) The each specimen of bony was preserved with embalming solution and kept at a temperature below 5°C for 5–7 days.[19]The soft-embalming solution was developed by the faculty of veterinary science, Chulalongkorn university which contained fixative agent, preservative agent, humectant agent, and antioxidant agent. The collected specimens were sent for biomechanical testing.

2.3 Histological and scanning electron microscope (SEM) evaluation.

All Specimens were evaluated an osteointegration process by 3D picture and histomorphology. Scanning electron microscope (SEM) was used to check a crack of total implant-bone interface. (Figure.4)

For histomorphology, all sections were cleaned up by normal saline and decalcified. After that the sections were placed in a slide to Hematoxylin and Eosin staining. The bone–implant interface was visualized using microscope.

2.4 Push-out testing

According to eight implants were used to evaluate ultimate interfacial strength. The implants were measured and adjusted an implant surface for perpendicular to vertical line. We adjust an alignment and measure by water level gauge (Figure.5A). Due to the testing process using the steel rod, it is necessary to ensure that the surface of the workpiece is in direct contact with the pressing rod to analyze the shear force accurately (Figure.5B).

The underlying receiver beneath the workpiece will have a circular opening at the centerline, equal to the size of the implant. This allows for movement of the implant against the bone when a load is applied. So, the bone will be counteracted by the underlying receiver beneath the workpiece. (Figure.6)

To evaluate the ultimate interfacial strength of the implants at the bone–implant interface, pull-out testing was performed using an Instron Model 1125 (Instron Corp, Canton, MA, USA).

The Instron machine was programmed at a cross-head speed of 5 mm/min. Ultimate interfacial strength (s) was calculated using the formula:

Ultimate interfacial strength = P / πdh

P was the ultimate pull-out load (N), d (mm) was the major diameter of the implant, and h (mm) was the length of the implant in the bone.

To ensure that a consistent compressive force is applied to the workpiece at all times, we will observe the graph of force increasing over the duration of the experiment. (Figure.7)

All animals tolerated surgical implantation well, and all animals regained full weight-bearing mobility within a three-day postoperative without any complications. At death, no signs of inflammation, gross infection, or tissue reaction were noted around the implant sites in any animals.

Histological and scanning electron microscope (SEM) evaluation.

Twelve weeks after implant placement, there are a new bone formation at bone-implant interface. Because the osteoblasts were found with blood vessel and multiple osteocytes were found within a lacuna. It is implied that a new bone formation was occurred surrounding at this area and no sign of infection or tissue necrosis (Figure.8). SEM investigate the interface between the implant and bone interface. There was no evidence of large bone detachment at all circumferential implants (Figure.9–10).

Mechanical result

Mechanical testing data is summarized in table 1 (Mean, median and standard deviation). The mean inter-face shear strength at 12 weeks post implantation is 11.32 MPa. Displacement of implant show in Figure.11 After implant was displaced, we found a residual bone on the surface of implant due to shear force break through cancellous bone (bone to bone interface) Figure.11 We calculated a residual bone compared to the total surface of implant. We found that implant with high shear strength is relate to low residual bone contact but not significant different (P < 0.05). Table.2

Specimen number	Push out force (N)	Ultimate interfacial strength (MPa)
1	7400	18.39
2	2250	5.59
3	6300	15.66
4	2600	6.46
5	4800	11.93
6	3600	8.95
7	5100	12.68
8	4400	10.93

Table.1 Push out test results, The implants were tested at a distraction rate of 0.5 mm/min.

Specimen number	Push out force (N)	Ultimate interfacial strength (MPa)	Residual bone contact/total surface	Percentage (%)
1	7400	18.39	42/250	16.8
2	2250	5.59	37.5/250	15
3	6300	15.66	86.5/250	34.6
4	2600	6.46	83/250	33.2
5	4800	11.93	45.12/250	18
6	3600	8.95	91.5/250	36.6
7	5100	12.68	36/250	14.4
8	4400	10.93	72/250	30

Table.2 Relationship between a shear strength and residual bone contact.

The objective of this investigation was to assess the shear resistance of HA-coated cpTi implants in a large animal model. These implants, with a large diameter and HA coating, were inserted into the intramedullary canal of the proximal femoral bone. The outcomes of this study provided evidence of successful osseointegration and impressive shear strength between the implant and bone interface in the intramedullary canal of the proximal femur in miniature pigs. These findings are consistent with previous studies that have reported positive outcomes with HA-coated implants. The HA coating on the implant facilitates osteointegration and enhances attachment strength. Furthermore, the utilization of a large animal model and intramedullary canal implantation in this investigation confer a significant advantage over previous studies that employed small animals and different types of implants. K.A. HING et al demonstrated an interfacial shear strength of 7.3 MPa in the femur of rabbits[12], while Stewart M reported a shear strength of 5.2 MPa.[13] Additionally, Ong, J.L. presented an interfacial shear strength of 3.4 MPa in a dog.[14] Large animal models offer a more representative representation of human bone and can yield more precise outcomes regarding implant stability and osseointegration, mirroring the situation in human hip arthroplasty. Our study revealed that the shear values were three times higher when compared to the previous investigation. Histological and SEM evaluation demonstrated the formation of new bone around the implant, with no signs of inflammation or tissue reaction. The push-out testing also revealed a shear strength of 11.32 MPa between the implant and bone interface, which is a crucial factor for ensuring implant stability and longevity. Joo L. Ong et al showed that pull-out strength is low due to the weak coating-substrate interfacial strength.[14] Moreover, Wang et al demonstrated a failure in the coating, with failure modes occurring within the coating lamellar splat layer.[16] Our study demonstrated that broken osteointegration occurred in two areas: the shear force occurred between the implant and bone interface, as well as between cancellous bone layers themselves (Figure.10). Specifically, we observed that shear force was present at the layers of osteointegration between the bone layer and the workpiece, as well as between cancellous bone layers themselves. In theory, the bonding strength of osteointegration (implant to bone interface) should be higher than the strength of trabecular bone (bone to bone interface). Therefore, given the high shear values observed in the experiment, we should anticipate shear forces to manifest between the bone and the implant. When we evaluated the amount of residual bone still attached to the workpiece in comparison to the total implant area, we discovered that a sample with a small amount of residual bone generally exhibited higher shear strength. This suggests that the osteointegration of this workpiece experienced more breakage at the implant-to-bone interface than at the bone-to-bone interface, although this difference is not statistically significant (Figure.10). Based on these results, we postulate that the shear strength at the implant and bone interface is higher than the shear strength at the bone and bone interface.

However, in the current scenario, the procedure employed for removing the femoral stem results in the disruption of osteointegration at the interface between the implant and bone, as well as bone-to-bone interaction.

It is imperative to acknowledge the existence of certain constraints within this study. Firstly, the sample size consisting of only three animals is relatively modest, thereby necessitating further investigations encompassing a larger sample size to corroborate these findings. Moreover, the long-term outcomes pertaining to shear strength were not demonstrated in this study.

In conclusions, this study demonstrates that HA-coated cpTi implants provide excellent shear strength and osseointegration in the proximal femur of miniature pigs. These findings showed a failure mode at bone-to-bone interface similar to femoral stem revision. However, further studies with a larger sample size and different bone sites are necessary to confirm these findings.

Acknowledgements

The authors would like to thank Dr. Nadhapat Bunnag from Chulalongkorn University, an expert in anesthesia for experimental animals, for her contributions to this research. Additionally, we extend our gratitude to Dr. choopet Nitsakulthong from Chulalongkorn university laboratory animal center for his assistance in taking care of the experimental animals.

Data availability statement

The data supporting this study’s finding are available from the corresponding author(C.V.)upon reasonable request.

Additional information

Correspondence and requests for materials should be address to C.V.

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No competing interests reported.

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You are reading this latest preprint version

Analysis of the shear strength of a hydroxyapatite (HA)-coated commercially pure titanium (cp-Ti) implant: A in vivo study in pigs

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Abstract

Figures

Introduction

Experimental and Technical design

2.1 implant materials

2.2 Implantation procedure

2.4 Push-out testing

Results

Mechanical result

Discussion

Declarations

References

Additional Declarations

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