Complications and Outcome of Bone Sarcoma Patients with Limb Salvage using Liquid Nitrogen-treated Bone for Reconstruction

Primary bone sarcomas are uncommon, accounting for <0.2% of malignant neoplasms with distinct incidence patterns and clinical behavior.^[1] Various age-specific histologic subtypes are reported with bimodal age distribution, with one peak seen in the adolescent and pediatric population while another occurs after 50 years of age.^[2] Treating bone sarcoma in the pediatric and adolescent population is challenging as the growing skeleton poses several changes, and they are more physically active than the adult population.^[3]

In the past, radical treatment like amputation was preferred in adolescents. However, with the advancement of chemotherapy regimens, modern implant design, reconstructive options, and surgical techniques, limb salvage has become the standard for bone sarcoma.^[4] However, multiple issues related to the adolescent and pediatric populations, such as mechanical failure, loosening, and durability, have also been reported.^[5,6]

Several methods of biological reconstruction have been documented in literature irradiated, autoclaved, pasteurised, and frozen.^[7–10] Extracorporeal radiotherapy has the disadvantage of taking bone out of the theatre, while pasteurization results in the loss of both biological and mechanical properties of bone.^[9,11] Liquid nitrogen-treated bone retains both osteoconductive and osteogenic properties.^[12] Liquid nitrogen recycled bone is advantageous in selected reconstruction patients because it provides anatomically matched grafts and restores the bone length with sufficient strength.^[10,13] Several studies documented better results with liquid nitrogen-treated bone reconstruction with the optimal bone union and functions, and it is the preferred reconstruction method in complicated cases.^[14,15] However, complications and technical difficulties with using liquid nitrogen, such as handling, fractures, nonunion, and wound-related problems, are well known.^[16]

In developing countries like Pakistan, liquid nitrogen-treated bone for limb salvage is an economical option. However, late presentation with sizeable soft tissue components and long length of bone involvement constitutes a significant concern resulting in major resection of involved bone, over which the effect of liquid nitrogen has still not tested.^[17] The objective of this study was to determine the complications and functional outcomes associated with limb salvage surgery using liquid nitrogen-treated bone.

This retrospective review was conducted in the Department of Surgical Oncology, Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan, after obtaining ethical approval from the institutional review board. Patients with primary malignant musculoskeletal tumors of the extremities who had undergone limb salvage surgery using a liquid nitrogen-treated bone graft from January 2018 to December 2020 with a minimum of 1-year follow-up were included in the study. Data were collected and entered on pre-designed forms.

All patients with limb sarcoma received neoadjuvant chemotherapy after initial presentation and workup. Patients with osteosarcoma underwent two cycles of Methotrexate, Doxorubicin, and Cisplatin (MAP) regimen neoadjuvant chemotherapy followed by surgery, then four cycles of adjuvant chemotherapy. Patients with Ewing sarcoma underwent six cycles of Vincristine, Doxorubicin, Cyclophosphamide alternating with Ifosfamide, Etoposide regimen neoadjuvant chemotherapy followed by surgery, then seven cycles of adjuvant chemotherapy.

We have used the free-frozen method of treating bone with liquid nitrogen as described by Tsuchiya et al.^[10] Tumor-bearing bone was frozen in the liquid nitrogen for 20 min, then at room temperature, and distilled in water for 10 and 15 min. The frozen bone autograft was used for reconstruction. Bone graft or cement was used for mechanical support when necessary. The frozen bone was reconstructed using locking plates or intramedullary rods. We had performed intercalary resection in 14 patients, and one patient of distal femur underwent osteoarticular resection. To increase vascularity, free vascularized fibula graft was used in two patients of distal femur sarcoma along with liquid nitrogen-treated bone in two patients. Immediately after the surgery, patients were allowed range of movement exercise. Partial weight-bearing was allowed 2 months after surgery, and weight protection was continued until sufficient callus formation at the host graft junction occurred.

Patients were followed regularly for wound and functional score assessment with musculoskeletal tumor society (MSTS) score and Toronto extremity scoring system for operated limbs. In addition, patients underwent an X-ray chest for pulmonary metastasis and a computerized tomography (CT) scan of the involved limb for any recurrence and to assess bone union every 4 months. Based on the radiological findings of the CT scan limb, auto grafts that were conserved were deemed as “survived,” and those that had been resorbed and were no longer functional were recorded as “died”. Survival of autografts was recorded using the Kaplan–Meier method with a 95% confidence interval.

Statistical Package for the Social Sciences software (SPSS^®) 23 was used for statistical analysis. Continuous variables as means ± Standard Deviation (SD) and analyzed using Student’s t-test. Categorical data as numbers and percentages and analyzed using χ² or Fisher’s exact test as appropriate in all the above statistical tools. P ≤ 0.05 was considered to be statistically significant.

Fifteen patients were reviewed; 11 (73%) were male and 4 (27%) were female. The mean age of patients was 13 years (range 7–22 years). The most common histopathology was osteosarcoma in 12 patients, while three had Ewing sarcoma. The distal femur was the common location for the oncological disease. It was seen in seven (46.7%) patients. Metastatic disease was not seen in any patients at the time of presentation.

Twelve (80%) patients were alive, while 3 (20%) died during the follow-up period. All of these patients had advanced disease with pulmonary metastasis. The mean follow-up of the remaining 12 patients was 19.83 ± 4.5 months.

Of the 12 surviving patients, in six patients, the liquid nitrogen grafts survived. Among those in whom the graft died, two patients developed a deep infection after surgery, one developed a recurrence, and three had a mechanical graft failure. Details are presented in Table 1. The mean survival of liquid nitrogen treated graft was 8.4 ± 2.09 (months) [Figure 1].

Figure 1:

Survival curve for liquid nitrogen graft with mean survival of liquid nitrogen treated graft in our study is 8.4±2.09 (months)

Eleven patients had a bone union (22 osteotomies), with the average union time being 6.9 ± 1.3 months. Four patients had non-union. Of these four (seven osteotomies), one patient underwent revised plating with bone graft to achieve union. Meanwhile, another patient presented with nonunion at the distal femur proximal site and was replaced with endoprosthesis reconstruction. The remaining two patients ended up in amputation.

Post-operative histopathology of the sample showed closed margins (<5 mm) in four patients. However, none of these patients had recurrence during this study. Nonetheless, among the other patients, three developed recurrence, and the average time to recurrence after tumor resection was 6.7 ± 1.3 months. Two patients had recurrence after distal femur reconstruction with liquid nitrogen-treated bone, while one had recurrence at the proximal tibia.

Eleven (73%) patients developed complications after liquid nitrogen treatment, with superficial wound problems occurring in 8 (53.3%) patients, deep infection in 3 (20%) patients, and foot drop and wrist drop in one patient each.

The average MSTS score in liquid nitrogen-treated reconstruction was 62.4 ± 7.9%, while the average Toronto extremity score was 59.6 ± 5.7%. The functional score was lower in the proximal tibia, having a score of 51.7 ± 3.1, while the distal tibia had the highest functional score of 66.7%.

The method of freezing bone with liquid nitrogen has been gaining popularity, especially in young individuals [Figure 2]. The outcome of liquid nitrogen-treated bone is better than allograft due to the lower rate of graft rejection.^[18] This study assessed the complications and functional outcomes associated with limb salvage surgery using liquid nitrogen-treated bone.

Figure 2:

Magnetic resonance imaging (MRI) axial (a) and coronal (b) views of 12-year-old male having osteosarcoma who underwent reconstruction with liquid nitrogen treated bone as shown in X-ray tibia anteroposterior (c) and lateral (d) views

We used the free frozen method of liquid nitrogen treatment, which increases the osteotomy sites, and chances of complication. Shinozaki et al. described the pedicle freezing method of liquid nitrogen bone treatment, which decreased osteotomy site and had a better union rate as compared to free freezing.^[19] The method of pedicle freezing is technically more difficult and data are limited regarding technical difficulties in using liquid nitrogen for pedicle freezing.^[20]

Li et al., in their study, have shown a good functional outcome of liquid nitrogen-treated bone reconstruction with MSTS score of more than 80% and better outcomes in terms of gait in their patients.^[21] On the contrary, Wisanuyotin et al. have documented low functional outcomes with an MSTS score of <70% with biological reconstructions.^[22] The MSTS score of our patient was low (<60%). This may be due to delayed presentation and extensive resection of bone length in our population.

Comparable to our findings of an osteotomy union rate of 76% and a length of bone resection more than 209 ± 57.3 mm, the literature review found that the functional outcome had a bone union rate of more than 90% with a resected length of the bone 138.4 ± 60.39 mm. This suggests that there are more chances for non-union and complications with increased resection length.^[15,23]

Therefore, to decrease the rate of non-union at the osteotomy site pedicle freezing method has been introduced which decreased the number of osteotomies and had better functional outcomes.^[19] The use of vascularized fibula along with cryotherapy has shown better outcomes.^[24] To increase the vascularity of bone, we have used vascularized fibula graft in two patients with Distal Femur Osteosarcoma numbered 11 and 12 in Table 1, which had shown early signs of union and decreased complications as compared to other patients.

Another method to increase bone union and decrease complications is to perform hemi cortical resection of diseased bone as described by Chen WM et al.^[25,26] With this resection, one cortex remains in continuity, resulting in better structural support and more chances of liquid nitrogentreated graft survival. However, this method is valid only for exophytic and small tumors. Furthermore, we did osteoarticular dissection in one patient with distal femur osteosarcoma and treated dissected bone and articular cartilage in liquid nitrogen. Higuchi T also has described knee preservation surgery using liquid nitrogen-treated bone with free freezing and pedicle method and documented the excellent functional outcome.^[27] However, in our series, the developed dissociation of the epiphysis from the shaft at the level of physis results in impingement of the shaft with the patella and poor functional outcome.

Recurrence was seen in three patients, occurring from the soft-tissue component. The proximal tibia was the most common site of recurrence, probably due to the lack of soft tissue coverage around the proximal tibia and large soft-tissue component, making resection more difficult. The recurrence rate was slightly higher than described in the literature, which may be due to the extensive disease and large soft-tissue component in our patients.^[13,21] Out of the 15 patients who underwent liquid nitrogen reconstruction in our series, 5 of them (33%) experienced distant metastasis. The lungs were the most commonly affected site. Similarly, Yoshihiro Arak’s study also reported a distant metastasis rate of over 26% in patients who underwent liquid nitrogen treatment.^[28] One of our patients had also developed metastasis in the contralateral tibia (solitary bone metastasis) along with pulmonary metastasis, which were rare findings not reported in the literature.

Skin sloughing and deep wound infection was the most common complication after liquid nitrogen reconstruction, which resulted in a delay in adjuvant chemotherapy, increased hospital stays, and poor outcomes. Skin sloughing was seen in more than 50% of our patients, which led to deep wound infection or required revision surgeries to cover the implant. Wound-related problems reported in the literature are comparable to our findings as one of the major complications after liquid nitrogentreated bone reconstruction.^[28,29] The absence of growth of immune cells and the hypothermic effect of liquid nitrogen on the surrounding structure may contribute to skin necrosis and infection. However, this effect of liquid nitrogen-treated sarcomas needs to be tested in future studies.^[30]

We have observed the death of three patients, making the survival of bone sarcoma <80%, which is comparable to other studies, showing 3-year survival of 79%.^[18] The weakest aspect of using liquid nitrogen bone grafts was that pathological examination was not performed, and the percentage of necrosis was not calculated. Because of this, the effectiveness of the preoperative chemotherapy regimen cannot be determined, which resulted in the use of the same chemotherapy regimen in adjuvant cycles.^[22] This may also result in an increase in recurrence and metastasis rate after liquid nitrogen reconstruction. This may also result in an increase in recurrence and metastasis rate after liquid nitrogen reconstruction. The limitation of this study was a small number of patients with a relatively shorter follow-up period. The small sample was due to the limited use of liquid nitrogen in only those patients in which adulttype mega prosthesis was not feasible. However, satisfactory reconstruction was achieved in all patients with minimal complications. The scope of our research encompasses a wide range of patients with varying medical conditions, including upper and lower limb injuries, as well as extra-articular and osteoarticular dissection. The distinct range of medical conditions may have an impact on the outcomes of our study.

Liquid nitrogen-treated autologous bone replantation is an alternate option for primary bone sarcomas in all age groups, especially in resourcelimited environments, where allografts and end prostheses are difficult to obtain. However, it is a safe and reliable biological reconstruction method with a high complication rate, low healing end of osteotomy, and non-satisfactory postoperative function on short-term follow-up. Further, several aspects of this technique have yet to be thoroughly evaluated but may be helpful to consider for future studies to better treat this aggressive disease.