A novel aragonite-based scaffold for osteochondral regeneration: early experience on human implants and technical developments

Injury, Int. J. Care Injured 47S6 (2016) S27–S32

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A novel aragonite-based scaffold for osteochondral regeneration: early experience on human implants and technical developments Elizaveta Kona, *, Dror Robinsonb, Peter Verdonkc, Matej Drobnicd, Jenel Mariano Patrascue, Oliver Dulicf, Gordon Gavrilovicg, Giuseppe Filardoa a

NanoBiotecnology Lab, I Clinic - Rizzoli Orthopaedic Institute, Bologna, Italy Department of Orthopedics, Hasharon Hospital affiliated with Tel Aviv University, Rabin Medical Center, Petah Tikwa, Israel Antwerp Orthopaedic Center, Monica Hospitals, Stevenslei, Deurne, Belgium and Department of Orthopaedic Surgery, Faculty of Medicine, Antwerp University, Wilrijkstraat, Edegem, Belgium d Department of Orthopedic Surgery, University Medical Centre Ljubljana, Slovenia e Spitalul Clinic Județean de Urgență “Pius Brînzeu” Timișoara Bulevardul Liviu Rebreanu, Timișoara, Romania f Kliniki Centar Vojvodina, Novi Sad, Serbia g The Institute for Orthopaedic Surgery “Banjica”, Beograd, Serbia b c

K E Y W O R D S

A B S T R A C T

Cartilage

Introduction: Chondral and osteochondral lesions represent a debilitating disease. Untreated lesions remain a risk factor for more extensive joint damage. The objective of this clinical study is to evaluate safety and early results of an aragonite-based scaffold used for osteochondral unit repair, by analysing both clinical outcome and MRI results, as well as the benefits of the procedure optimization through novel tapered shaped implants. Methods: A crystalline aragonite bi-phasic scaffold was implanted in patients affected by focal chondralosteochondral knee lesions of the condyle and trochlea. Twenty-one patients (17 men, 4 women with a mean age 2 of 31.0 ± 8.6 years) without severe OA received tapered shaped implants for the treatment of 2.5 ± 1.7 cm sized defects. The control group consisted of 76 patients selected according to the same criteria from a database of patients who previously underwent implantation of cylindrical-shaped implants. The clinical outcome of all patients was evaluated with the IKDC subjective score, the Lysholm score, and all 5 KOOS subscales administered preoperatively and at 6 and 12 months after surgery, while MRI evaluation was performed at the 12 month follow-up. Results: A statistically significant improvement in all clinical scores was documented both in the tapered implants and the cylindrical group. No difference could be detected in the comparison between the improvement obtained with the two implant types, neither in the clinical nor in imaging evaluations. A difference could be detected instead in terms of revision rate, which was lower in the tapered implant group with no implant removal – 0% vs 8/ 76–10.5% failures in the cylindrical implants. Conclusions: This study highlighted both safety and potential of a novel aragonite-based scaffold for the treatment of chondral and osteochondral lesions in humans. A tapered shape relative to the cylindrical shaped implant design, improved the scaffold’s safety profile. Tapered scaffolds maintain the clinical improvement observed in cylindrical implants while reducing the postoperative risk of revision surgery. This aragonite-based implant was associated with a significant clinical improvement at the 12 month follow-up. Moreover, MRI findings revealed graft integration with good bone and cartilage formation. © 2016 Elsevier Ltd. All rights reserved.

Osteochondral repair Biphasic Aragonite Scaffold Knee

Introduction Chondral and osteochondral lesions represent a debilitating disease, which if left untreated would progress to more extensive joint * Corresponding author at: Dr. Elizaveta Kon, NABI Laboratory – I Clinic, Rizzoli Orthopaedic Institute, Via Di Barbiano, 1/10 - 40136 Bologna. Tel: +39 051 6366567; Fax: +39 051 583789. E-mail address: [email protected] (E. Kon).

0020-1383 / © 2016 Elsevier Ltd. All rights reserved.

damage eventually leading to the development of osteoarthritis [1]. Clinical and research activities are ongoing in an effort to improve outcomes and over the years different surgical techniques have been suggested [2]. Among these, regenerative scaffold-based procedures are emerging as a potential therapeutic option, with an increasing number of publications every year on in vitro, preclinical animal studies and clinical applications [3,4], but none has shown tissue healing with biomechanical properties that match the physiological condition.

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Lately, the awareness of the involvement of subchondral bone in many of these lesions, resulted in the development of cell-free treatment strategies focused on the entire osteochondral unit [5–7], and currently heterogeneous scaffolds have been proposed that combine distinct but integrated layers corresponding to the cartilage and bone regions to regenerate both components of the osteochondral unit to restore the articular surface [8–11]. Most scaffolds attempt to regenerate the articular cartilage by implantation of soft biomaterials to act as a cartilage surrogate that is supposed to allow surface reconstitution. Recent literature described a different approach to target osteochondral regeneration: an aragonitebased bi-phasic scaffold, which showed promise in the preclinical model [12,13]. To date, only a preliminary report in humans has been published, with positive results at 24 months for the treatment of a patient affected by a chronic posttraumatic cartilage lesion of the knee [14]. However, prior to wide range clinical application, it is important to document both safety and feasibility of this procedure, which is based on the implant of a scaffold relying on a press-fit fixation. Considering that sufficient attachment and graft stability in the early period are essential for the successful outcome of any technique, since an insufficient graft fixation may facilitate the detachment of the transplanted biomaterial and lead to treatment failure [15], we focused on the stability evaluation of this procedure, which has been first developed with cylindrical implants and recently optimized through a new shape for tapered implants. To document the early postoperative adherence rate in humans, invasive approaches are not appropriate due to patient safety concerns and disruption of the primary stability. Thus, magnetic resonance imaging (MRI) is useful as a non-invasive technique for the analysis of the morphological status of cartilage defects and the repair tissue throughout the postoperative period [16,17]. In the current work, we evaluated both early clinical results as well as MRI imaging of a pilot group of patients undergoing implantation of the tapered aragonite-based scaffold with the objective to compare the results of treatment to a historical control of patients who previously underwent cylindrical scaffold implantation. Materials and methods Scaffold characteristics and implantation procedure The implant (Agili-C™, CartiHeal, Israel) consists of a natural crystalline aragonite, derived from corals, to which hyaluronic acid (HA) is added. The natural aragonite, possess a nano-rough structure as well as interconnecting porosity that allows to stimulate cell adhesion and proliferation as well as matrix production [18]. A square grid pattern of 2 mm deep drilled channels is made in the top part, using Bungard CCD, a CNC drilling and routing machine and an appropriate drill-bit as described in US patent application 20120177702 & 20120189669. HA is added to the top part of the implant. A preliminary preclinical study in a goat model showed the benefit of a scaffold configuration with mechanical modification through micro-drilling of the top layer and HA added to it [12]; the early potential of both bone and cartilage formation at 6 months was confirmed by a subsequent study of the same model at 12-month follow-up with scaffold degradation and osteochondral regeneration [13]. Thus this scaffold configuration was used and developed in the shape of cylinders for the treatment of both chondral and osteochondral defects in humans. Recently, a tapered version of the implants, with an angle of 2 degrees from the longitudinal axis, has been designed to improve the press-fit implantation (Figure 1). Before the clinical application, an extensive purification process is performed to treat and remove trapped particles, debris and organic remnants, and the implants are sterilized by 25 kGy gamma radiation. The surgical procedure is performed with the patient under anesthesia and in the supine position. A pneumatic tourniquet is placed on the proximal thigh and a mini arthrotomy medial or lateral

Fig. 1. Tapered aragonite-based implant.

parapatellar approach is used to expose the lesions. The defect is then prepared using proprietary surgical toolset (Cartiheal, Israel). A perpendicular aligner is used to center the lesions and place a Kwire, which is used to correctly position a drill sleeve where a motorized drill is inserted to prepare the defect up to the desired depth. A reamer is then inserted to ensure the correct depth is obtained and a shaper is introduced to finalize the lesion with the correct wall inclination. Then the peripheral cartilage is trimmed and, after debris removal, the tapered implant is inserted manually and subsequently gently impacted to a position 2 mm below the surface of the articular cartilage through a silicone-covered tamper. The stability of the transplant is tested by cyclic bending of the knee while the graft is under direct vision, both before and after tourniquet removal. The rehabilitation program includes toe-touch weight bearing (with no significant amount of weight) using crutches for 4 weeks, with increasing partial weight bearing reaching full weight bearing after 6 weeks. During the first 48 hours cryotherapy in conjunction with a continuous-passive-motion (CPM) device are applied and carried on for 3 weeks, together with active assisted range-ofmotion exercises. Quadriceps isometric sets and electrostimulation are initiated immediately after surgery. Stationary cycling is introduced at 4 weeks, when the patient reaches knee flexion of 100°. Hydrotherapy is advised immediately after suture removal. Resistance musclestrengthening exercises are initiated after the third month. Outdoor cycling activity and skiing are allowed not earlier than 6 months after the operation, while repetitive joint impact activities are allowed after 1 year. Patients’ selection and evaluation The study involves patients enrolled, treated, and prospectively followed after Hospital Ethics Committee and Internal Review Board approvals. Inclusion criteria for the selection of patients for this pilot evaluation were: patients affected by focal chondral-osteochondral knee lesions ICRS grade III-IV of the condyle and trochlea, with Kellgren Lawrence lower than 3 and age younger than 50 years. Exclusion criteria were: concomitant ICRS grade 4 lesions on the patella or tibial plateau, multiple lesions and evidence of severe joint degeneration at the pre-operative X-Ray of Kellgren Lawrence >2, and patients with non-corrected misalignment or instability of the knee ( patients who

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presented with an anterior cruciate ligament-ACL lesion at the time of surgery underwent the associated surgical procedure of ACL reconstruction in the same surgical session with the osteochondral grafting). Patients with infectious, neoplastic, metabolic and inflammatory pathologies were also excluded from the study, as well as those not able to comply with the required post-operative rehabilitation regimen. Overall, 21 patients were enrolled and treated: 17 were men and 4 women, with a mean age of 31.0 ± 8.6 years and a mean BMI of 26.2 ± 3.4. The location of the defects was the following: 11 medial femoral condyles, 4 lateral femoral condyles and 6 trochleas. The average size of the defects was 2.5 ± 1.7 cm2. Eight patients were surgically treated for the first time, whereas 13 patients had undergone previous surgeries: 7 ACL reconstructions, 7 meniscectomies, 2 microfracturing, 1 shaving and 1 debridement of chondral lesions, and 1 soft tissue removal. In one patient combined ACL reconstruction and meniscectomy were performed. A control group of 76 patients was selected according to the same exclusion criteria from a database of cylindrical implants: 62 were men and 14 women, with a mean age of 31.7 ± 7.8 years and a mean BMI of 25.1 ± 3.4. The sites of the defects were the following: 45 medial femoral condyles, 25 lateral femoral condyles, 6 trochleae, and the average size of the defects was 1.7 ± 1.0 cm2. Thirty patients were surgically treated for the first time, 46 patients had undergone previous surgeries, and 26 also underwent combined surgery. The two groups were homogeneous except for combined surgery (higher in the control group; P = 0.006), site (more trochleae in the tapered group; P = 0.032), and a tendency for bigger lesions in the tapered group (P = 0.069). Nonetheless, these aspects were found not to influence the results thus allowing the group comparison both in terms of clinical and imaging outcomes. The clinical outcome of all patients was evaluated with the IKDC subjective score, the Lysholm score, and all 5 KOOS subscales administered preoperatively and at 6 and 12 months after surgery. Failures were documented as well. Nineteen patients of the tapered implants group also underwent imaging evaluation at 12 months follow-up. Examinations were carried out with a 1.5 or 3 T MRI. The MOCART MRI scoring system was used as a reference for the evaluation of the graft [19]; however, since this score was originally designed as an ACI evaluation system and focuses on variables mainly related to the cartilage layer, the following parameters were used in the analysis: defect fill, cartilage interface, bone interface, surface, structure, signal intensity, subchondral bone plate, chondral osteophytes, bone marrow edema, subchondral bone, and effusion.

Fig. 2. IKDC subjective score improvement in cylindrical and tapered implants.

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Statistical analysis All continuous data were expressed in terms of the mean and the standard deviation of the mean, the categorical data were expressed as frequency and percentages. The Kolmogorov Smirnov test was performed to test normality of continuous variables. The Repeated Measures General Linear Model (GLM) with Sidak test for multiple comparisons was performed to assess the differences at different follow-up times. The ANOVA test was performed to assess the between groups differences of continuous, normally distributed and homoscedastic data, the Mann Whitney test was used otherwise. The Spearman rank Correlation was used to assess correlation between continuous data. Fisher Chi square test was performed to investigate the relationships between dichotomous variables. Pearson Chi square test evaluated by Exact Methods for small samples was performed to investigate the relationships between grouping variables. For all tests p < 0.05 was considered significant. All statistical analysis was performed using SPSS v.19.0 (IBM Corp., Armonk, NY, USA). Results A statistically significant improvement in all clinical scores was documented. In particular, in the tapered implant group the IKDC subjective score increased from 36.5 ± 14.2 to 58.9 ± 18.5 at 6 months and 63.2 ± 18.0 at 12 months (P < 0.005) (Figure 2). Similarly, the Lysholm score increased from 54.8 ± 18.5 to 70.9 ± 16.5 at 6 months and 75.6 ± 17.2 at to 12 months (P < 0.005) (Figure 3). An increase was also recorded in all KOOS subscales (Table 1). MRI evaluation performed in 19/21 patients (2 unavailable for imaging evaluation) showed 84% patients with >75% defect fill, 84% with a cartilage interface complete or with demarcating border but no defect visible, 89% with complete bone interface, 84% with smooth intact surface or damaged <50%, 84% with homogeneous structure, 84% with normal/nearly normal signal intensity, 95% with intact subchondral bone plate, 63% with no bone marrow edema, and 89% with intact subchondral bone. However, in 1 case a large effusion and no osteophytes formation was observed. In the control group of cylindrical implants the IKDC subjective score increased from 46.9 ± 15.9 to 58.1 ± 19.1 at 6 months and 70.0 ± 22.3 at 12 months (P < 0.005) (Figure 2). Similarly, the Lysholm score increased from 62.1 ± 18.8 to 74.1 ± 19.6 at 6 months and 79.8 ± 19.4 at 12 months (P < 0.005) (Figure 3). An increase was also recorded in all KOOS subscales (Table 1). MRI evaluation performed in 68/76 patients

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Fig. 3. Lysholm score improvement in cylindrical and tapered implants.

(8 underwent implant removal and thus 12 months MRI evaluation couldn’t be performed) showed 84% patients with >75% defect fill, 90% with a cartilage interface complete or with demarcating border but no defect visible, 78% with complete bone interface, 79% with smooth intact surface or damaged <50%, 78% with homogeneous structure, 93% with normal/nearly normal signal intensity, 79% with intact subchondral bone plate, 59% with no bone marrow edema, 78% with intact subchondral bone, 15% with a large effusion and only 2 cases with osteophytes. The comparison of the two groups underlined a difference in the preoperative scores which were lower in the tapered group (IKDC subjective score P = 0.011, KOOS Pain P = 0.033, KOOS ADL P = 0.004, KOOS QoL P = 0.082, Lysholm P = 0.075). However, no difference could be detected in the comparison between the score improvement obtained with the two implant types, neither in the clinical and imaging evaluations. However, a difference could be detected instead in terms of revision rate which was lower in the tapered implant group (no implant removal – 0% vs 8–10.5% failures in the cylindrical implants) (Figure 4). Discussion Biological based therapies for the treatment of bone and cartilage lesions have been evolving as a result of all the advances made in regenerative medicine [20–26]. The main finding of this study is that implantation of the aragonitebased scaffold allowed to obtain a significant clinical improvement. The improvement following implantation of the scaffold was

Table 1. KOOS subscales improvement from basal level and 6–12 month follow-ups KOOS

Basal

6 months

12 months

P: basal-12 m

Cylindrical Implants Symptoms Pain ADL Sport QoL

63.8 ± 17.6 60.3 ± 16.0 69.6 ± 18.1 33.4 ± 22.6 28.7 ± 16.7

75.7 ± 21.4 78.9 ± 19.2 84.0 ± 18.5 51.6 ± 32.7 46.4 ± 25.8

80.5 ± 20.3 82.2 ± 20.1 87.5 ± 19.0 66.3 ± 29.9 55.9 ± 29.6

<0.005 <0.005 <0.005 <0.005 <0.005

Tapered Implants Symptoms Pain ADL Sport QoL

55.6 ± 23.4 52.0 ± 14.8 56.4 ± 18.0 29.1 ± 24.3 22.0 ± 16.7

73.6 ± 19.1 79.0 ± 16.4 86.5 ± 12.4 54.0 ± 27.6 48.2 ± 29.7

79.8 ± 17.6 81.4 ± 13.9 86.6 ± 13.5 59.5 ± 30.2 50.6 ± 27.7

<0.005 <0.005 <0.005 <0.005 <0.005

documented by both MRI imaging and clinical results. While efficacy is similar with both designs, safety is increased with the optimized tapered implants. Aragonite or hydroxyapatite based scaffolds, derived from corals’ exoskeletons are commonly used as bone graft substitute and bone void filler in orthopaedics, cranial and maxillo-facial surgery, as well as periodontal and plastic surgery [12,18], being a natural material similar to human bone, including its three-dimensional structure and crystalline form of calcium carbonate (CaCO3). Aragonite-based scaffold derived from Porites coral presents osteoconductive and osteogenesis properties and allow a gradual degradation leaving a functional bone tissue in its place [27,28]. However, this material without the proper treatments is lacking the ability to induce joint surface repair [29]. HA is a safe and optimal intra-articular lubricant [30–34]. In the current study, this biodegradable and biocompatible natural polymer has been impregnated onto the top phase of the scaffold to lubricate the articular surface during the immediate post implantation period. Previously, promising results in terms of both bone and cartilage formation in the goat model at 6 month were noted using this design [12], later confirmed with good osteochondral regeneration at 12 months [13]. This scaffold was initially developed for human application with a cylindrical shape which resembles mosaicplasty, a traditional technique for the treatment of articular surface lesions, targeting the cartilage layer by capitalizing on bone-to-bone healing because the mature cartilaginous tissue has limited healing potential and troublesome integration with the surrounding cartilage. While good longterm results have been reported with mosaicplasty, donor site morbidity remains a major concern of autologous osteochondral transplantation [35,36], and osteochondral scaffolds have been advocated to avoid the need for autologous material and associated morbidity. This aragonite-based scaffold takes advantage of positive aspects of the mosaicplasty approach while avoiding some negative ones. Nonetheless, in terms of surgical technique this osteochondral scaffold still faces some of the same challenges as mosaicplasty. This technique relies on the healing capability of live cancellous bone to anchor osteochondral graft tissue, with a successful result exhibiting graft fixation capable of weight bearing while the donor graft integrates with the cancellous host bone. Initial graft fixation is achieved through a press-fit, where adequate congruency is vital for primary stability [37]. Any graft movement due to inadequate fixation strength or penetration of synovial fluid prior to bone ingrowth would therefore be detrimental. Inadequate fixation strength may also lead to an

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Pre-op MRI

Implantation

Post op X-ray

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12 months MRI

Fig. 4. 20 year old man treated for a large trochlea osteochondral defect. IKDC improved from 60 to 86 and overall KOOS from 46 to 90 at 12 months of follow-up.

interruption of vascular formation, with further deterioration of the graft anchorage in the end jeopardizing the treatment outcome [38]. As a result, initial graft load-bearing capacity is primarily dependent on the fixation strength provided by the press-fit. In order to further protect the implant and prevent graft mobilization, following mosaicplasty, patients are advised to remain non–weight bearing for several weeks after surgery. This recommendation makes rehabilitation rather long and doesn’t fully guarantee graft stability provided by the press-fit fixation in the early postoperative period. In fact, substantial deterioration of short-term fixation strength of the mosaicplasty plugs has been documented from the immediate post-operative state [38]. Such a reduction in short-term graft load bearing capacity may pose a threat to the surgically established articular surface congruency and blood vessels formed during the early stages of the healing response. Similarly, in order to provide a successful outcome this aragonitebased implant needs to remain stable in the treatment site not just postoperatively but also in the following phases during full weightbearing and physical activities. To this regard, the aragonite-based scaffold is implanted in a recessed position that does not extend to the level of the articular surface and protect the implant. Moreover, improvement of primary stability with the use of a tapered scaffold allows a more advantageous result. In the current study, the early results of this scaffold were analysed using both imaging and clinical evaluations. Non-invasive evaluations were preferred to invasive ones. Arthroscopy is unsuitable for routine follow-up due its invasive nature and associated risks, and biopsies may damage the healing tissue due to disruption of primary stability and may not be representative of the entire treated area. In contrast, MRI has become the method of choice for non-invasive postoperative monitoring of osteochondral lesions and repair tissues [39,40]. Thus, we used MRI scans for the analysis of the graft, and 19 out of 21 patients treated with the novel tapered implants were available for the evaluation at 12 months after implantation, showing overall good results in all the parameters assessed. In particular, even though this follow-up may still be considered an early time point in terms of osteochondral tissue regeneration, most of the implants presented good filling of the lesion and integration of the graft with both good bone and cartilage formation and a clear demarcation of the two regenerating tissues. In this phase of ongoing healing process the presence of two differentiated tissues without any presence of “bone step-in” is very promising for the good restoration of the normal osteochondral unit structure. A correlation between clinical and imaging outcomes could not be identified, probably due to the low number of cases evaluated and the well documented difficulties on the overall literature in correlating clinical and imaging findings [41]. Moreover, a comparison of the imaging results could not be performed with the cylindrical implant group, since this presented some cases of implant removal that didn’t allow the MRI evaluation of the scaffold at 12 months. However, MRI confirmed the good healing potential of this scaffold also in the cylindrical group, and the clinical

results allowed to complete the analysis of the outcome offered by the two groups. In fact, the clinical evaluation showed, besides overall positive findings, the advantages of using the tapered version of the implants. Despite some unfavourable conditions due to larger lesions and worst clinical basal level, the tapered implants confirmed a similar significant clinical improvement, with further advantages in terms of no need for any implant removal. The tapered sides at an angle of two degrees represent a minor shape change of substantially cylindrical plugs, but may still explain the improved results with respect to the unmodified implants of otherwise identical composition and structure. These may be explained by some advantages of the tapered shape, which is self-centering the implant into the defect site. This helps avoiding possible damages related to a not perfectly perpendicular plug introduction. Moreover, great press fit forces are created even when using relatively small tamping force during insertion, further reducing the risk of implant damage. These aspects may be reflective of a better preservation of the scaffold integrity during insertion, with the maintenance of a better press-fit and consequently an enhanced incorporation and in the end an improvement in terms of adverse events and failures while offering the same benefits of the more widely documented cylindrical implants in terms of clinical outcome. Limitations of this study are the short-term follow-up and the retrospective nature of the groups comparison. Nonetheless, all patients were prospectively followed and the 12 months results still offered important findings. Even though such an early clinical evaluation is not useful for determining the success rate of the procedure; the scores obtained documented a significant improvement with respect to the preoperative level. This short-term observation period provided important results in terms of safety healing potential of this scaffold, documenting the benefits of the optimized tapered implants. The scaffold ability to induce osteochondral tissue without the need of autologous cells makes it attractive both from a practical and commercial standpoint, since it could be used as an off-the-shelf graft in a one-step surgical procedure, and from a surgical standpoint, since due to the possibility to safely implant it under minimally invasive conditions. Previous animal studies highlighted the good regenerative potential of this aragonite-based osteochondral scaffold in promoting both bone and hyaline cartilage tissue restoration by itself [12,13], and this pilot evaluation confirmed the potential of this material implanted in humans with a press-fit technique, with a significant post-operative improvement of the patients evaluated at 12 months. Thus, while further prospective evaluation is necessary to determine the clinical and morphological outcome at longer follow-up, the results of this study showed that these tapered aragonite-based scaffolds offered promising results in terms of osteochondral healing potential and a satisfactory clinical improvement for the treatment of articular surface lesions.

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