Injectable Scaffold with Microfracture using the Autologous Matrix-Induced Chondrogenesis (AMIC) Technique: A Prospective Cohort Study

Introduction Autologous matrix-induced chondrogenesis (AMIC) is a one-step surgical cartilage repair procedure involving the insertion of a scaffold into the chondral defect after microfracture. BST-CarGel [Smith and Nephew, Watford, England] is an injectable chitosan-based scaffold which can more easily fill defects with irregular shapes and be used to treat vertical or roof chondral lesions. The study aims to evaluate the clinical outcomes of knee cartilage repair with microfracture surgery and BST-CarGel using the AMIC technique for a minimum of two years. Materials and methods A prospective study of patients undergoing cartilage repair with microfracture surgery and BST-CarGel at our institution from 2016 to 2019 was performed. Clinical outcomes were determined using the Lysholm Knee Scoring System and Knee Injury and Osteoarthritis Outcome Score (KOOS). These questionnaires were administered before the surgery and at a minimum of two years after surgery. Results A total of 21 patients were identified and recruited into the study. 31 cartilage defects were seen and treated in 21 knees. These included horizontal lesions (e.g., trochlear, lateral tibial plateau), vertical lesions (e.g., medial femoral condyle, lateral femoral condyle) and inverted lesions (e.g., patella). No complications or reoperations were seen in our study population. For the average duration of follow-up of 42.5±8.55 months, there was an average improvement in Lysholm score of 25.8±18.6 and an average improvement in KOOS score of 22.5±15.0. Conclusion BST-CarGel with microfracture surgery using the AMIC technique is a safe and effective treatment for cartilage defects in the short to medium term.


INTRODUCTION
Chondral lesions of the knee are a common injury seen in the younger population. Widuchowski et al reported chondral lesions in 60% of a cohort of over 25,000 knee arthroscopies with an average age of 39 years old 1 . Due to the avascular, aneural, and immune-privileged nature of hyaline cartilage, the regenerative potential of cartilage after an injury is limited. The quest to repair cartilage in one-stage minimally invasive surgery is one of the holy grails in orthopaedic surgery.
Cartilage repair is recommended for symptomatic lesions in patients less than 55 years old. The techniques of cartilage repair include marrow stimulation, cell-based techniques such as autologous chondrocyte implantation, osteochondral autologous transplantation and autologous matrix-induced chondrogenesis (AMIC).
AMIC is a one-step surgical cartilage repair procedure involving the insertion of a scaffold into the chondral defect that provides a structure that mechanically stabilises the super clot and stimulates chondrogenic differentiation 2 . BST-CarGel [Smith and Nephew, Watford, England] is an injectable chitosan-based scaffold. Chitosan is a deacetylate derivative from chitin, which is found in exoskeletons of shellfish and insects 3 . It is biocompatible, biodegradable and shown to be a suitable scaffold for chondrocyte proliferation and differentiation [4][5][6] .
The study aims to evaluate the clinical outcomes of knee cartilage repair with microfracture surgery and BST-CarGel using the AMIC technique for a minimum of two years of follow-up.

MATERIALS AND METHODS
A prospective study of patients undergoing cartilage repair with microfracture fracture and BST-CarGel [Smith and Nephew, Watford, England] at our institution from 2016 to 2019 was performed. Clinical outcomes were determined using two clinically validated patient-reported questionnaires: Lysholm Knee Scoring System 7 and Knee Injury and Osteoarthritis Outcome Score (KOOS) 8 . These questionnaires were administered before surgery and at a minimum of two years after surgery.
For the operative technique, diagnostic arthroscopy of the knee joint was first performed with the use of a four-lead gravity fluid bag. Standard anterolateral and anteromedial portals were created adjacent to the patellar tendon. Using a 30° scope via the anterolateral portal, the cartilage injuries were assessed for the severity of lesion and graded according to the Outterbridge grading system, and other concomitant soft tissue injuries were inspected (Fig. 1). Where appropriate, concurrent procedures, such as meniscal repair and ligament reconstruction were performed in the same setting, before cartilage repair.
BST-CarGel was prepared according to the techniques described in the manufacturer's product guide: The buffer solution was mixed with the chitosan solution and left for 10 minutes. A total of 5ml of autologous blood drawn via venepuncture was injected into the prepared buffer-chitosan mixture. This BST-CarGel blood-buffer-chitosan mix was drawn into an application syringe and ready to be applied to the cartilage lesion.
The cartilage defect site was debrided using shavers and curettes. Loose chondral tissue was excised to create stable vertical walls surrounding the defect. The bed of the defect was prepared with the preservation of the subchondral plate. Microfracture was then performed with arthroscopic microfracture awls to a depth of about 4mm, with each lesion created 3 or 4mm apart throughout the surface of the cartilage defect (Fig. 2).
The fluid was drained from the joint and dry arthroscopy was performed (Fig. 3). The knee surface and cartilage lesion were dried using suction and patties. The injectable scaffold was delivered as a viscous gel to horizontal lesions (eg. trochlear, lateral tibial plateau), vertical lesions (eg. medial femoral condyle, lateral femoral condyle) and inverted lesions (eg. patella) (Fig. 4). The scaffold was left to solidify within the defect for 5-10 minutes (Fig. 5). The knee was then mobilised, and wet arthroscopy repeated to ensure the stability of the scaffold in a fluid-filled environment.
For post-operative care, the rehabilitation range of motion protocol was dependent on the location of the lesion. For lesions of the condyles or plateau, patients were placed in a knee brace and motion restricted from 0° to 90° to limit deep flexion. For patellofemoral lesions, knee flexion was limited in the first three weeks, with the range of motion gradually increased to 90° flexion by 6 weeks. All patients were kept non-weightbearing for the first three weeks and gradually progressed to protected weightbearing (< 50% of body weight) over the next three weeks. This was to protect the microfracture clots and scaffold augmentation from dislodgement, which may occur on deep flexion or weightbearing.
Informed consent was obtained from patients before the collection of patient data. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 1983.

RESULTS
The demographics of our study population and characteristics of cartilage lesions are summarised in Table I. A total of 21 patients were identified and recruited into the study. 85.7% were male, with an average age of 41.2 years old (range = 17-57 years old). In 65% of patients, the cartilage defect was in the right knee. An open approach was employed in five patients, with a mini-open approach used in one patient. An arthroscopic approach was used in 15 patients.
The average size of each defect was 3.36 ± 3.54cm 2 (range = 0.06-12.25cm 2 ). The lateral meniscal repair was performed in the same setting on three patients. The medial meniscal repair was done on three patients, with an additional anterior cruciate ligament repair performed on one patient. Lateral patellar retinacular release, medial patellofemoral ligament repair and high tibial osteotomy were performed on one patient each. No complications or reoperations were seen in our study population.
The patient-reported outcomes are summarised in Table II One patient reported reduced Lysholm score by 1 (preoperative score = 79; post-operative score = 78) and reduced KOOS score by 9.7 (pre-operative score = 85.7; postoperative score = 76) on 41-month follow-up. The patient underwent a concurrent lateral patellar retinacular release and had three areas of defects: 4cm 2 trochlear defect, 0.8cm 2 medial femoral condylar defect, 0.5cm 2 central patellar defect. A second patient reported reduced Lysholm score by 6 (pre-operative score = 79; post-operative score = 73) on 38-month follow-up. This second patient had two areas of defects: 1cm 2 lateral femoral condylar defect, 0.35cm 2 trochlear defect. A 0.5cm 2 medial femoral condylar defect with Grade 2 changes was also noted but did not undergo repair.

DISCUSSION
AMIC or "microfracture plus" techniques for cartilage repair are gaining popularity due to their attractiveness as a singlestage surgery. The evidence shows superior tissue repair in radiological and histological studies and improved outcomes in clinical studies 9,10 . Chondral lesions treated with the AMIC technique also had a nearly normal morphologic appearance 11 . Our study has shown that microfracture with BST-CarGel is an effective procedure in providing symptomatic relief in patients with cartilage defects in the knee.
Microfracture is the first-line option for lesions of <2.5cm 2,12 . It involves perforation of the exposed subchondral plate within the cartilage defect to form a "super clot" of marrow contents with mesenchymal stem cells to differentiate into  fibrocartilage repair tissue 13 . It has been a mainstay in cartilage repair due to its effectiveness in providing symptomatic relief in the short-to medium-term 14 . However, the high fibrocartilage content in the repair tissue leads to poor long-term functional outcomes 15 and the dislodgement of the microfracture clot may account for a lack of defect fill seen in post-operative MRIs 16 .
ACI is a two-stage procedure that harvests cartilage from regions with reduced weight-bearing requirements for expansion to chondrocytes in a lab; the chondrocytes are then implanted in the second surgery 17 . Its effectiveness lies in the ability to provide repair of the defect with more hyaline cartilage. Although it is more effective than other methods for lesions > 4cm, it is a very costly technique 12,[17][18][19] .
AMIC is an enhancement of the microfracture technique, where the scaffold inserted into the chondral defect adds stability to the super-clot, anchoring it within the defect and reducing the risk of dislodgement 2 . In addition, scaffolds,    The use of solid scaffolds like Chondro-Gide and Hylofast in cartilage repair often requires additional fixation when applied to the defect, usually with fibrin glue. Whyte et al described a technique for the use of Hyalofast in cartilage repair. The Hyalofast solid scaffold was first cut to the appropriate size before being delivered and applied to the defect under dry arthroscopy; fibrin glue was recommended as an adjunct should more stability be required 24 . The benefit of an injectable scaffold is that it allows ease of application via arthroscopy. Being viscous in nature, these scaffolds can fill the defect with gradual solidification. They can more easily fill larger defects with irregular shapes and can be used to treat vertical or roof chondral lesions.
BST-CarGel is an injectable chitosan-based scaffold. It is mixed with autologous whole peripheral blood before injection into the defect. This scaffold-blood mixture impedes repair clot retraction and increases adhesiveness, thus stabilising the super-clot within the defect 25  Cartifill, an injectable porcine collagen scaffold, has also shown good outcomes. Kim et al conducted a multicentre, randomised, parallel-group trial comparing microfracture with Cartifill vs microfracture alone, looking at patientreported outcomes at baseline, 12 months and 24 months post-intervention. There was a significantly higher odds of improvement in pain score in the Cartifill group as compared to microfracture alone. The MRI outcomes using modified MOCART, 50% defect filling and the ratio of repair tissueto-reference cartilage were also significantly higher in the Cartifill group 29 .
Our post-operative functional outcomes are consistent with the studies on the use of enhanced microfracture techniques. Our outcomes at an average follow-up of 42.5 months postoperatively show that 90.5% (19/21) had improvement in patient-reported outcome scores. The two patients that did not have improvements had multiple cartilage lesions. This is also the first study using this scaffold in an Asian population. The option of using an injectable non-porcine derived scaffold provides us additional treatment options when treating patients who may have religious or ethical concerns about using porcine tissue. This is an important consideration in Asia.
Steinwachs et al described the use of BST-CarGel scaffold augmentation of a microfracture procedure with the use of dry arthroscopy. They recommended positioning the knee such that the lesion was in a horizontal position before application to avoid the gel scaffold dripping off the surface 30 . In our cohort, we have used the injectable gel scaffold in hard-to-treat positions such as vertical or even inverted lesions, so long as the injectable gel scaffold is of the right viscosity when applied. The meticulous practice of keeping the scaffold warm allows us to achieve the viscosity for stable application onto vertical or inverted lesions.
There were several limitations of our study. Firstly, as this study was conducted at a single institution by a single surgeon, only a small sample size was available. There was also a large range of defect sizes seen in our population. This, in addition to the fact that several patients underwent concurrent procedures, may have affected their reported outcomes. We were unable to stratify these factors to correlate with their outcomes due to the small sample size. However, this method enabled us to control for surgeondependent factors such as technical skill and familiarity with using BST-CarGel. There is potential for future studies to capture a large population size by using a standardised technique, and to differentiate outcomes based on defect size and concurrent procedures.

CONCLUSION
BST-CarGel with microfracture surgery using the AMIC technique is a safe and effective treatment for cartilage defects in the short to medium term.

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.