Comparing the Rate of Dissolution of Two Commercially Available Synthetic Bone Graft Substitutes

Abstract

This study characterized the dissolution properties of two commercially available bone substitutes: (1) A calcium sulfate (CaS)/brushite/β-tricalcium phosphate (TCP) graft containing 75% CaS and 25% calcium phosphate; and (2) a CaS/hydroxyapatite (HA) bone graft substitute composed of 60% CaS and 40% HA. Graft material was cast into pellets (4.8 mm outer diameter × 3.2 mm). Each pellet was placed into a fritted thimble and weighed before being placed into 200 mL of deionized water. The pellets were removed from the water on days 1, 2, 3, 4, 6, 8, 14, 18, or until no longer visible. The mass and volume of each pellet were calculated at each timepoint to determine the rate of dissolution. Analysis of variance was performed on all data. Statistical significance was defined as p < 0.05. The CaS/HA pellets were completely dissolved after day 8, while the CaS/brushite/β-TCP pellets remained until day 18. The CaS/brushite/β-TCP pellets had significantly more mass and volume at days 1, 2, 3, 4, 6, and 8 timepoints. The CaS/brushite/β-TCP pellets lost 46% less mass and 53% less volume over the first 4 days as compared to CaS/HA pellets. The CaS/brushite/β-TCP pellets had a rough, porous texture, while the CaS/HA pellets had a smooth outer surface. Overall the CaS/brushite/β-TCP pellets dissolved approximately twice as slowly as the CaS/HA pellets in vitro. As these in vitro findings might have in vivo implications, further clinical data are required to further confirm and establish the optimal synthetic bone substitute strategy or antibiotic delivery carrier.

Keywords

bone graft substitute - dissolution - resorption

In the United States, approximately 500,000 bone grafting procedures are performed every year.[1] Among all clinically available grafts, autologous bone is still considered the gold standard.[1] [2] However, there are disadvantages to autograft, such as donor site morbidity and limited supply.[3] The use of allograft is another viable option and offers the advantage of avoiding donor site morbidity. However, there are still risks of disease transmission with allografts, even though the methods of preparing and processing these grafts have improved greatly in recent years.[4] As the population ages, there is a growing demand for spinal fusion, revision arthroplasty, joint fusion, and other orthopaedic procedures requiring a bone graft.[5] [6] [7] As a result of the shortage of donor tissue and little chance of meeting the demands of an aging population, scalable alternatives to bone grafting are needed.[2] To address this growing issue, many synthetic bone graft substitutes have been developed over the past several decades.[8]

The development of new orthobiologic materials including ceramic-based synthetic bone substitutes alternatively based on hydroxyapatite (HA) and tricalcium phosphates (TCP), have been widely used in clinical practice to aid in the management of bony defects.[8] Due to growing demand, and the wide variety of synthetic bone substitutes developed, comparisons among commercially available grafts are needed.[9] When creating a synthetic bone graft, it is ideal to have a graft that is biocompatible and will support new bone growth. The rate of resorption of synthetic bone substitutes affects mechanical stability and varies depending on the graft material composition used.[10] Ideally, bony ingrowth would occur at the same rate as graft degradation, thus maintaining mechanical stability. Calcium sulfate (CaS), HA, and β-tricalcium phosphate (β-TCP) are three materials commonly used in bone substitutes, and each has a different rate of dissolution. CaS resorbs most rapidly (1–3 months), followed by β-TCP (4–5 months) and HA (up to 3 years).[11] By combining these materials in different ratios, it is possible to adjust the rate and degree of absorption in order to optimize mechanical strength.

Therefore, the objective of this study was to characterize the dissolution properties of two commercially available composite bone graft materials: a CaS/brushite/β-TCP graft containing 75% CaS and 25% calcium phosphate (PRO-DENSE™, Stryker, Memphis, TN) and a CaS/HA bone graft substitute composed of 60% CaS and 40% HA (CERAMENT, Bonesupport, Lund, Sweden). Specifically, this study will report on the average mass and volume of bone substitutes remaining at various timepoints over an 18-day period in order to compare their relative rate of resorption.

Materials and Methods

Materials

Two clinically available synthetic bone substitutes tested in this study were (1) PRO-DENSE™—a CaS/brushite/β-TCP composed of composite containing 75% CaS and 25% calcium phosphate (brushite and granular TCP); and (2) CERAMENT™—a CaS/HA bone graft substitute composed of 60% CaS and 40% HA. The PRO-DENSE™ and CERAMENT™ products used in this study are listed in [Table 1]. Calcium sulfate dihydrate (CSD) was used as reference material. The CSD is the same CaS component found in the hardened PRO-DENSE material. It is pure CaS and conforms to the ASTM 2224 standard specification for surgical implants. All products were unexpired and sourced from commercially available inventory.

Table 1 Product evaluated

Part number	Lot number	Description
87SR0050	1690129	PRO-DENSE™ 5 mL
87SR0050	1677316	PRO-DENSE™ 5 mL
87SR0050	1684020	PRO-DENSE™ 5 mL
8004000	MLOT0382	CEREMENT™ 5 mL
8004000	MLOT0431	CEREMENT™ 5 mL
8004000	MLOT0452	CEREMENT™ 5 mL

Pellet Formation

Graft material was mixed per the manufacturer's instructions and cast into pellets (4.8 mm outer diameter × 3.2 mm) 3 minutes from the start of mixing. Pellets were cured for a minimum of 8 hours at room temperature (20 ± 10 °C), demolded, and oven-dried at 40 ± 2 °C for a minimum of 5 hours.

Mass

Each pellet was placed into a fritted thimble and weighed before being placed into 200 mL of deionized water within a 250-mL Nalgene polymer bottle. Utilization of fritted glass thimbles allows removal of the pellets at test intervals without damaging the pellets. Each bottle was placed in a 37 °C water bath and the water in each bottle was replaced daily. Five fritted thimbles (per lot) were removed from the water on days 1, 2, 3, 4, 6, 8, 14, and 18, and oven-dried at 37 °C for a minimum of 5 hours. If no pellet mass remained, the oven drying process was not performed. The mass of each pellet before and after dissolution was calculated by subtracting the mass of the corresponding fritted thimble. The pellet mass remaining at each timepoint was calculated as follows:

Volume

After weighing, pellets were removed from the fritted thimble and measured using calipers. Testing continued until no visible pellet volume remained in the thimble. Pellet volume was determined by approximating the pellet as a cylinder. The initial pellet volume was determined by measuring five pellets from each lot prior to dissolution testing. The remaining pellet volume at each timepoint was calculated as follows:

Statistical Analysis

Statistical analyses were performed using Minitab 16 (Minitab Inc., State College, PA). Analysis of variance was performed on all data. Statistical significance was defined as p < 0.05.

Results

Mass

The average amount of mass remaining at each dissolution timepoint is shown in [Fig. 1]. Raw data are summarized in [Table 2]. The CaS/HA pellets were completely dissolved after day 8, while the CaS/brushite/β-TCP pellets remained until day 18. As a point of reference, pure CSD pellets were completely dissolved by day 7. There was significantly more CaS/brushite/β-TCP graft material at days 1, 2, 3, 4, 6, and 8 timepoints compared to CaS/HA material.

Table 2 Average percentage mass remaining following dissolution

Percentage mass remaining	Day	0	1	2	3	4	6	8	14	18
PRO-DENSE™	Average	100	88.9	78.3	72.3	65.7	54.5	42.9	16.1	14.3
	SD	–	1.3	1.8	2.8	1.8	3.4	3.9	2.2	1.6
CERAMENT™	Average	100	74.9	55.0	44.9	36.7	23.3	16.0	–	–
	SD	–	1.8	2.8	7.4	4.3	6.0	3.6	–	–

Abbreviation: SD, standard deviation.

Fig. 1 Percent mass remaining throughout the dissolution period. *Denotes significance versus CERAMENT (p < 0.05). n = 5 per timepoint. CSD, calcium sulfate dihydrate. *Denotes significance versus CERAMENT (p < 0.05).

The rate of dissolution of CaS/brushite/β-TCP and CaS/HA pellets is shown in [Fig. 2]. The rate of dissolution was determined using the slope of the curves over the first 4 days of this accelerated test. There was a 46% reduction in mass loss per unit time for the CaS/brushite/β-TCP pellets compared to CaS/HA. Comparing these results to dissolution data of a pure CaS material, CaS/brushite/β-TCP has a 52% slower dissolution rate versus pure CaS, while CaS/HA has a 12% slower rate.

Fig. 2 Rate of mass loss throughout the study period. CSD, calcium sulfate dihydrate.

Volume

The average pellet volume at each dissolution timepoint is shown in [Fig. 3]. The raw data are summarized in [Table 3]. Significantly more CaS/brushite/β-TCP pellet graft volume was observed at days 1, 2, 3, 4, 6, and 8 timepoints. The CaS/HA pellets and CaS/brushite/β-TCP pellets were completely dissolved by days 8 and 18, respectively. Analysis of the rate of volume loss, calculated by the slope of the curves over the pellet lifetime, shows that the CaS/HA graft (y = − 0.13) dissolved more than twice as fast as CaS/brushite/β-TCP (y = − 0.06).

Table 3 Average percentage volume remaining following dissolution

Percentage mass remaining	Day	0	1	2	3	4	6	8	14	18
PRO-DENSE™	Average	100	93.0	88.3	84.3	79.6	65.8	43.1	9.1	0.0
	SD	–	2.4	3.7	3.3	6.1	7.1	9.1	4.5	0.0
CERAMENT™	Average	100	82.0	58.5	44.9	33.7	9.9	0.0	–	–
	SD	–	4.5	5.6	8.6	6.4	4.2	0.0	–	–

Abbreviation: SD, standard deviation.

Fig. 3 Percent volume remaining throughout the dissolution period. *Denotes significance versus CERAMENT (p < 0.05).

Representative images of the pellets at each dissolution timepoint are shown in [Fig. 4]. The CaS/HA pellets had no remaining structural integrity by day 8 based on visual inspection, with a breakdown of the cylindrical pellet shape, and thus no measurable volume despite having residual mass. Similarly, a small amount of CaS/brushite/β-TCP mass remained at day 18 according to measurements, however, the material was visually indistinguishable with no defined edges, reflecting the completion of dissolution. Therefore, no volume calculation could be performed. The overall size of the CaS/HA pellet is significantly reduced even at early timepoints. Additionally, the surfaces of the pellets are noticeably different, with the CaS/brushite/β-TCP pellets having a rough, porous appearance and the CaS/HA pellets having a smooth outer surface.

Fig. 4 PRO-DENSE and CERAMENT pellets. Representative images of pellet volumes at each dissolution timepoint.

Discussion

Although autografts are still considered the gold standard for bone grafting, their widespread usage is limited by donor site morbidity and supply. Over 500,000 bone grafting procedures are performed annually in the United States and more than 2 million worldwide.[12] [13] As the population ages, demand for joint replacement, spinal fusion, joint fusion, fracture repair, and other orthopaedic procedures and bone grafting continues to increase. Consequently, orthobiologic materials, including ceramic-based synthetic bone substitutes utilizing HA and TCPs, are becoming more widely used in clinical practice to aid in the management of bony defects. A wide variety of synthetic bone substitutes have been developed over the past decade, and comparison among available grafts is needed. As the rate of resorption of synthetic bone substitutes has an effect on mechanical stability and varies depending on the graft material composition used, dissolution kinetics remains a topic of interest.

Both PRO-DENSE and CERAMENT are commercially available composite bone graft materials comprising CaS and slower-resorbing calcium phosphate. PRO-DENSE consists of approximately 75% CaS and 25% brushite and β-TCP. CERAMENT comprises 60% CaS and 40% HA. Despite PRO-DENSE having a greater amount of CaS than CERAMENT, the dissolution rate was significantly slower.

Calcium silicate hydrate, also known as plaster of Paris, was first used as a bone graft in the late 1800s.[2] [14] [15] It is weakly osteoconductive, inexpensive, and easily prepared, making it an increasingly popular component in synthetic bone substitutes.[11] CSH reacts with water to form CSD and is commonly referred to as CaS in the context of surgical implants. However, CaS alone has a rapid resorption rate and is not commonly used in isolation.[2] [11] CaS generally degrades within 1 to 3 months after insertion, therefore it resorbs faster than bone deposition and has weak internal strength.[11] [16] When combined with other synthetic bone substitutes, though, it can be made more durable.[2] PRO-DENSE utilizes a mixture of CaS, β-TCP, and brushite. β-TCP ceramics are considered the “gold standard” of synthetic bone grafts.[15] [17] β-TCP has been used as a bone substitute for over 25 years, and the material of this graft is the most similar in chemical composition to human bone.[1] The resorption rate of β-TCP is highly dependent on porosity and volume implanted. Dense β-TCP configurations can take 1 to 2 years to fully resorb, while highly porous scaffolds and granules may resorb in 4 to 5 months.[11] [18] The porous structure may facilitate the colonization of osteogenic cells and nutrients via enhanced capillary ingrowth.[11] While the overall resorption remains somewhat variable, it is slower compared to CaS.[11]

CERAMENT is a bioresorbable synthetic bone graft substitute that combines CaS with HA. HA has a composition similar to bone, making it highly biocompatible.[15] Additionally, HA is osteoconductive,[19] allowing it to conduct tissue growth for bone regeneration.[19] However, HA is relatively resistant to chemical and biological degradation, and HA particles can remain at the site for over 3 years after implantation before fully resorbing.[20] [21] [22] The slow resorption of HA allows time for gradual bone ingrowth and cell colonization within the graft material.[20] However, the extended retention period of HA particles could theoretically impede later bone remodeling after stability is achieved, as they occupy space that new bone growth could utilize.[1] [11] The implications of this long-term clinical bone healing require further study. In this in vitro analysis, CERAMENT dissolved more rapidly than the PRO-DENSE graft, suggesting the CaS component degraded faster, while HA particles persisted. Further details on the specific dissolution and degradation properties of the individual components of these composite grafts would aid the interpretation of these results. Additional in vivo analyses are needed to determine the optimal characteristics and resorption kinetics of synthetic bone substitute materials for effective bone regeneration.

The dissolution kinetics of the CaS/brushite/β-TCP material have shown that early dissolution of the CaS material exposes a porous matrix of fine brushite crystals and larger TCP granules. This self-formed porous scaffold is visually apparent in the CaS/brushite/β-TCP pellets at each dissolution timepoint, and absent from the CaS/HA pellets ([Fig. 4]). As CaS is dissolved from the CaS/brushite/β-TCP surface, the finely dispersed brushite crystals of the remaining scaffold form a diffusion barrier that slows the rate of CaS hydrolysis from the remaining bulk material. The slower dissolution is believed to contribute to the overall two-times slower rate of volume loss of the CaS/brushite/β-TCP pellets compared to CaS/HA pellets, despite a larger compositional percentage of CaS in the respective products. While the CaS/brushite/β-TCP material showed a slower dissolution rate compared to the CaS/HA material in this in vitro study, it is important to consider whether the duration of graft persistence is adequate for bone healing clinically. Both materials were fully resorbed by 18 days in this accelerated in vitro test method. However, bone healing, especially in compromised patients, can take considerably longer. As both products are marketed as resorbable cement, they may not provide mechanical support long enough for bone ingrowth before dissolving away. This is a potential concern, as premature dissolution could lead to fracture subsidence in weight-bearing applications. For example, in depression fractures of the tibial plateau being managed with bone graft, premature graft dissolution could result in loss of fracture reduction. Further study is warranted to determine whether the dissolution kinetics of both the CaS/brushite/β-TCP and CaS/HA materials provide adequate persistence to facilitate bone healing across various clinical indications and patient populations. Differences observed in this in vitro study may not directly translate to performance in vivo.

The composition and setting reaction of these synthetic bone grafts contribute to their dissolution properties over time. The formation process of these synthetic bone grafts utilizes the crystallization of CaS to form an initial solid construct. Specifically, the CaS powder is mixed with a liquid component, triggering dissolution and reprecipitation of CSD crystals and unreacted CaS crystals that interlock around the brushite, β-TCP, or HA particulate also in the mixture. This crystallization process allows the initial graft to solidify into a rigid structure within minutes. However, while the calcium phosphate components are osteoconductive and provide longer-term stability, they are not the main participants in the self-setting crystallization reaction. Thus, as the CaS crystal lattice dissolves over time, these secondary components may become unsupported matrix particles if the CaS dissolves too quickly. By day 18 of this study, the fully dissolved CaS phase was no longer able to maintain macrostructure or volume around the TCP and HA, despite the residual particulates themselves being insoluble in water over this time period. This explains the absence of measurable volume despite some remaining mass. In contrast, some synthetic bone grafts are preformed into a solid porous structure without reliance on self-setting chemistry. These grafts will retain their structure over longer durations as they resorb through mainly cell-mediated processes rather than dissolution, maintaining space-filling capacity over years instead of weeks. Determining the ideal graft persistence and mode of resorption to balance healing time with bone ingrowth remains an area requiring further optimization and study.

There are several limitations to this study. This study does not provide a comprehensive assessment of the many available synthetic bone graft materials currently available. Rather this provides a limited evaluation of the dissolution kinetics of two commercially available grafts. Additionally, this study was performed in vitro and does not provide an in vivo assessment of graft performance. Therefore, the correlation between the findings of this study and possible clinical implications remains unknown. Further clinical data on the performance of synthetic bone grafts is required to establish the optimal synthetic bone graft composition.

Conclusion

Overall, the CaS/brushite/β-TCP graft dissolved approximately twice as slowly as the CaS/HA bone graft substitute in vitro. The dissolution profile of the CaS/HA more closely resembles that of a pure CaS material than a slower-resorbing bone graft. As these in vitro findings might have in vivo implications, further clinical data are required to confirm and establish the optimal synthetic bone substitute strategy or antibiotic-eluting graft.

Conflict of Interest

N.S.P. is a paid consultant for Stryker Corporation. M.S. and J.F. are paid employees of Stryker Corporation.

Authors' Contributions

K.M.: interpretation of data, drafting the paper and revising it critically, approval of the submitted and final versions

M.S.: research design, acquisition, analysis and interpretation of data, approval of the submitted and final versions

I.P.: interpretation of data, drafting the paper and revising it critically, approval of the submitted and final versions

P.J.R.: interpretation of data, drafting the paper and revising it critically, approval of the submitted and final versions

J.F.: research design, acquisition, analysis, and interpretation of data, approval of the submitted and final versions

N.S.P.: research design, critical manuscript revisions, supervision, approval of the submitted and final versions

All authors have read and approved the final submitted manuscript.

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Publication History

Received: 12 May 2023

Accepted: 04 September 2024

Accepted Manuscript online:
05 September 2024

Article published online:
11 October 2024