Correction of secondary deformity after Nuss procedure for pectus excavatum by means of cultured autologous cartilage cell injection.

INTRODUCTION: For some cases of pectus excavatum, ideal chest shape cannot be achieved solely by performing the Nuss procedure. This manuscript presents a case where the residual deformity following Nuss was corrected using injection-transplantation of cultured autologous chondrocytes. PRESENTATION OF CASE: The treatment was performed for an 18-year-old male, who sought improvement of his chest shape after previously undergoing the Nuss procedure. A 1cm(2) auricular cartilage piece was harvested from his ear. Chondrocytes were isolated from the cartilage piece and were cultured. The cultured chondrocytes were processed into gel form and were injection-transplanted to the deformed region of the patient's chest. The grafted chondrocytes consolidated in one month, presenting elasticity equivalent to ordinary costal cartilage. The patient's chest remains in an optimal shape after a one-year postoperative follow up. DISCUSSION: Secondary correction of the chest deformity after previous operation for pectus excavatum is often tricky, because of the possible adhesion of the lungs or pericardium with the thoracic wall. Transplantation of cultured autologous chondrocytes does not require intra-thoracic maneuvers, and so is less invasive than other surgical interventions. Hence, priority can be placed, in some cases, on the chondrocyte transplantation rather than the re-correction of the thorax with the Nuss procedure or Ravitch procedure.
CONCLUSION: Transplantation of cultured autologous chondrocytes is recommended as a useful option for secondary correction of chest deformity after the Nuss procedure.

Introduction

Pectus excavatum is a congenital deformity characterized by concavity of the chest, occurring with frequency of 0.1–0.8 cases per 100 persons [1]. Currently, the Nuss procedure―in which the concave part of the thorax is elevated by using metal bars [2-4] is the standard treatment method for this congenital deformity and is less invasive than conventional methods [5]. However, it does not necessarily produce satisfactory results for all patients. There are cases, where unevenness remains despite the corrective bars or where recurrence gradually develops after their removal. This manuscript introduces a case in which recurrent chest deformity from pectus excavatum was corrected by transplantation of cell-engineered autologous chondrocytes.

Cell-engineering technique

Material for cell culture and transplantation was prepared by Yanaga’s method as follows [6-9]. A 1 cm2 auricular cartilage piece was harvested from the patient's auricular choncha (Fig. 1A), The piece was minced and rinsed with phosphate-buffered saline supplemented with penicillin G, streptomycin sulfate, and amphotericin B. The sample was then treated in 0.3% collagenase (Worthington Biochemical, Freehold, N.J.), spun with a stirrer at 37 °C for four hours, and filtered through a 100 μm-pore size cell-strainer (BD Bioscience, San Jose, CA) thereby isolating the chondrocytes. Chondrocytes were seeded at a cell density of 1 × 103 cells/cm2 and cultured in DMEM medium supplemented with 10% autoserum, FGF-2 (5∼10 ng/μl, FIBRAST®, Kaken Pharmaceuticals, Tokyo), penicillin G (400 unit/μl), streptomycin sulfate (1 mg/μl) and amphotericin B (2.5 μg/μl, Invitrogen Co., UK). The chondrocytes in their initial stage of growth are shown in Fig. 1B. The cultured cells were seeded three times on previous lineages [7] During three-passage culture of six weeks, the chondrocytes multiplied by 1000 times and presented a multilayered structure (Fig. 1C). The multilayered chondrocytes were seeded onto the subculture cells and gel-formed graft material was obtained as the final product, to be injected into patients.

Fig. 1

(A) Harvested auricular cartilage (B) chondrocytes of the primary passage, immediately isolated from the harvested cartilage (C) chondrocytes present multi-layered structure in multilayer culture system.

Clinical case

An 18-year-old boy visited the authors' institute seeking improvement of the appearance of his chest. He had previously undergone the Nuss procedure at the age of thirteen and the bar was removed when he was 16. After its removal deformity of the thorax gradually recurred, presenting the appearance shown in Fig. 2 at the age of 18. The options of a second Nuss procedure, reshaping of deformed cartilages with the open approach, cartilage graft from the hypochondrial region of the thorax, and injection-transplantation of cultured autologous chondrocytes were presented to the patient together with detailed explanations of their advantages and disadvantages. The patient requested the transplantation of the cultured autologous chondrocytes. Hence, after obtaining permission of the institutional review board of the authors' institute and informed consent from the patient and his family, harvesting and processing of the patient's auricular cartilage piece into an injectable cartilage gel were performed according to the previously described method. Thereafter, transplantation of the cultured cartilage was performed under general anesthesia. The skin above the deformed region was marked with dye, and subdermal dissection of this region was conducted through a small incision at the midline, at the layer just above the perichondrium of the xiphoid region (Fig. 3). A total of 60 μl of gel—with a cell density of 1 to 5 × 107 cells per μl—was injected into the dissected pocket made by the dissection, until the injected region became flat. To keep the grafted cartilage in an appropriate position, a sponge was applied to the overlying chest skin and continued for a month. By this time the transplanted cartilage had consolidated and had a firm elastic consistancy. One year after the transplantation, the chest shape retains an optimal shape, with no decrease of volume throughout the postoperative observation period (Fig. 4). Computer tomographic imaging taken at six months postoperatively revealed consolidation of the grafted cartilage (Fig. 5).

Fig. 2

Preoperative condition.

Fig. 3

Cultured chondrocytes were injected to the concave region (Blue marking area).

Fig. 4

Postoperative shape of the thorax at one year after operation.

Fig. 5

(Above) Preoperative computed tomographic image. (Below) A computer tomographic image at 5 months after the operation. Injected cartilage cells have consolidate (dotted circle), presenting with partial ossification (arrow).

Discussion

The Nuss procedure for pectus excavatum often fails to achieve full correction particularly of the hypochondrial region since the diaphragm interrupts positioning of correction the bars. Furthermore some cases develop recurrence of concavity once the correction bars are removed [10]. Treatment options for secondary correction for these deformities include a re-trial of the Nuss procedure, direct reshaping of deformed costal cartilages, and augmentation of the concave region by transplanting cartilage harvested from other regions of the thorax [11]. However, a secondary Nuss procedure is often risky, because of adhesion between the lung or pericardium and the thoracic wall. The latter two methods, reshaping and cartilage transplantation, are invasive and require additional incisions adding to the cosmetic deformity .Hence, we sought an alternative treatment involving cultured autologous auricular cartilage grafting.

There is no risk of injuring thoracic organs with cultured-chondrocyte grafting, because it can be implemented without intra-thoracic maneuvers. Furthermore, the method can be performed without making long scars on the chest. Instead, it only requires a small one centimeter long post auricular incision to harvest the auricular cartilage.

The disadvantage is that it takes several weeks to prepare the injectable material but this is compensated for by its reduced invasiveness.

A further theoretical advantage of our method is that it can also be applied to cases of severe deformity. For such cases cartilage transplantation from other sites on the thorax is not possible, because of insufficient donor cartilage. By contrast with cultured cartilage, even when the deformity is severe and requires a substantial graft, correction can be achieved by performing several cycles of transplantation. Once harvested, the chondrocytes can be frozen and preserved to be used for several cycles of treatment, if necessary.

Whether or not grafted cartilage remains permanently stable is unknown at the present stage. In our previous experiences augmenting noses, mandibles, and ears with cultured autologous chondrocytes using our technique, neo-cartilage has maintained its original shape with follow-up ranging from one to five years [10,11]. In the present case, the patient's chest retains optimal shape after one-year and we propose further follow-up to evaluate long-term results of the treatment.

Often in the treatment of pectus excavatum, as with other surgical procedures, patients are disappointed when an expected outcome is not achieved with the initial surgery and they are reluctant to agree to another invasive treatment for secondary correction. Grafting cultured autologous neo-cartilage presents a good alternative for such conditions, because of its minor invasiveness. Therefore, we recommend this method as a useful option for correction of secondary deformity after the Nuss procedure.

Conflict of interest

The authors have no financial interest to declare in relation to the content of this article. No external funding was received.

Source of funding

The present research was sponsored by Kagawa University hospital which has no involvement with any of following issues.

# Collection, analysis and interpretation of data.

# Writing or organizing the manuscript.

# Decision to submit the manuscript for publication.

Ethical approval

Ethical approve was obtained by the ethical committee of Kagawa University.

The registry number is Heisei 20-12.

Consent

The patient's privacy is secured, because the manuscript and its images don't carry any information by which the patient can be identified.

Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.

Author contribution

Motoki Tamai: performance of the treatment.

Tomohisa Nagasao: composition of the manuscript.

Hiroko Yanaga: culture of the chondrocytes.

Yusuke Hamamoto: asststance in surgical procedures.

Tetsukuni Kogure: care-taker of the patient during admission to the hospital.

Yoshio Tanaka: general director and supervisor of the study.

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