Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: A systematic review and meta-analysis.

BACKGROUND: Treatment of nerve injuries proves to be a worldwide clinical challenge. Vascularized nerve grafts are suggested to be a promising alternative for bridging a nerve gap to the current gold standard, an autologous non-vascularized nerve graft. However, there is no adequate clinical evidence for the beneficial effect of vascularized nerve grafts and they are still disputed in clinical practice.
OBJECTIVE: To systematically review whether vascularized nerve grafts give a superior nerve recovery compared to non-vascularized nerve autografts regarding histological and electrophysiological outcomes in animal models.
MATERIAL AND METHODS: PubMed and Embase were systematically searched. The inclusion criteria were as follows: 1) the study was an original full paper which presented unique data; 2) a clear comparison between a vascularized and a non-vascularized autologous nerve transfer was made; 3) the population study were animals of all genders and ages. A standardized mean difference and 95% confidence intervals for each comparison was calculated to estimate the overall effect. Subgroup analyses were conducted on graft length, species and time frames.
RESULTS: Fourteen articles were included in this review and all were included in the meta-analyses. A vascularized nerve graft resulted in a significantly larger diameter, higher nerve conduction velocity and axonal count compared to an autologous non-vascularized nerve graft. However, during sensitivity analysis the effect on axonal count disappeared. No significant difference was observed in muscle weight.
CONCLUSION: Treating a nerve gap with a vascularized graft results in superior nerve recovery compared to non-vascularized nerve autografts in terms of axon count, diameter and nerve conduction velocity. No difference in muscle weight was seen. However, this conclusion needs to be taken with some caution due to the inherent limitations of this meta-analysis. We recommend future studies to be performed under conditions more closely resembling human circumstances and to use long nerve defects.

Introduction

Treatment of nerve injuries proves to be a worldwide clinical challenge. Even though adequately treated, affected patients may suffer from chronic pain or lasting motor and sensory deficits [1]. For clinical situations in which it is necessary to bridge a nerve gap and when a tensionless coaptation is not possible, the current gold standard is an autologous non-vascularized (conventional) nerve graft. Treatment with a nerve graft always has a worse nerve recovery compared to primary coaptation, due to two anastomosis sides which increases the surface that needs to regenerate, ischemia of the graft and frequently a poor wound bed [2].

To improve the outcome after nerve repair with conventional nerve autografts the blood supply can be taken along with the nerve graft, the so-called vascularized nerve graft. Where a complete neurovascular bundle, which includes a nerve, its artery and its venae comitantes is harvested. The graft is placed in the middle of the nerve gap and the transected ends are sutured to the graft ends. Grafted nerves need considerable energy to regenerate and to maintain function. This energy is delivered by the intraneural vascular system, which is connected to extrinsic vessels. A vascularized graft is believed to restore these extrinsic neural blood vessels. Other theoretical reasons are: 1) the maintenance of vascularization promotes clearance of myelin debris, which results in faster remyelination; 2) the reduction of intraneural fibrosis as a result of ischemia eases axonal regeneration; 3) faster reinnervation reduces muscle atrophy [3-5].

There is no adequate clinical evidence of beneficial effect of vascularized nerve grafts except several case reports and case series [6-12]. These reported a superior two-point discrimination, sharp-blunt discrimination and warm-cold discrimination. The use of a vascularized nerve graft was first reported in 1976 by Taylor and Ham. They used 24 cm of the superficial radial nerve attached to the radial artery to reconstruct a median nerve defect [13].

Since the first publication by Taylor and Ham, many experimental studies in animal models have been reported. Vascularized nerve grafts have been successfully attempted in rats, rabbits, dogs, and other species to develop a model that is feasible, straightforward, reliable, and reproducible [14].

Nowadays, the use of vascularized nerve grafts is still debated in clinical practice because of several reasons: 1) the concern of a more significant donor site morbidity compared to conventional nerve autografts; 2) the lack of clinical evidence indicating the superiority of a vascularized nerve graft; 3) the difficulty to set up a controlled trial, due to the high heterogeneity of patients as well as nerve defects. For these reasons vascularized nerve grafts are no part of current clinical guidelines. Most institutions, including ours, use autologous nerve (cable) grafts as first option for long peripheral nerve gaps. If not enough autologous nerve is present, processed human allografts are used as second best [15-17]. However, both autologous and allografts lack vascularity.

Therefore, a systematic review and meta-analysis of animal models was conducted to investigate whether vascularized nerve grafts show a superior nerve recovery compared to non-vascularized nerve autografts regarding histological and electrophysiological factors.

Material and methods

Research protocol

This systematic review protocol was defined in advance and registered in an international database (PROSPERO, registration number CRD42020184363).

Search strategy

A systematic search has been performed in the PubMed (Medline) and Embase (OVID) databases to identify all original articles. The search included studies up to 26th of May 2020. Search terms included ‘nerve transfer’, ‘nerve graft’, ‘vascularized’ and ‘vascularization’ and their synonyms in abstract and title fields (for the complete search strategy, see S1 Table). The SYRCLE search filters to identify all animal studies were used [18, 19]. Duplicates were taken out using Endnote (Clarivate Analytics, Pennsylvania, USA). Two authors (BOB and TDJ) independently screened all titles and abstracts for their relevance utilizing predetermined inclusion and exclusion criteria. A reference- and citation check of the remaining studies was conducted manually to acquire potentially missed relevant articles. Afterward, the full text of the relevant articles was screened for final selection. Contradictory judgments were resolved by consensus discussion. No language or date restrictions were applied.

Inclusion and exclusion criteria

Articles were included when 1) the study was an original full paper which presented unique data; 2) a clear comparison between a vascularized and a non-vascularized autologous nerve transfer was made; 3) the population study were animals (all species) of all genders and ages; 4) the study investigated the effects of vascularized nerve grafts on: axonal count, diameter, nerve conduction velocity and muscle weight. No language or publication date restrictions were applied.

Critical appraisal

All included studies were appraised using the SYRCLE’s tool for assessing the risk of bias for animal studies [20]. This appraisal was done by two authors (BOB and TDJ) independently and subsequently merged by consensus. All criteria were scored a “yes” indicating a low risk of bias or a “no” indicating high risk of bias or a “?” indicating an unknown risk of bias. Baseline characteristics were: weight, age and race. Selective outcome reporting was determined by establishing if all outcome measures mentioned in Material and methods were reported in the Results section as well. To compensate for judging a lot of items as “unclear risk of bias” due to highly inadequate reporting of experimental details on animals, methods and materials, we included two items. The first item was reporting on any measure of randomization and the second item was reporting on any measure of blinding. Here a “yes” signifies reported and a “no” means unreported.

Data extraction

Data were in duplicate extracted from the selected studies by two authors (BOB and TDJ). The descriptive data included: publication year, first author’s name, studied species, gender, total number of animals, total grafts, studied nerve, studied muscle, graft length and time points. For the meta-analysis, the mean, sd and n of the following outcomes were extracted for axonal count, diameter, nerve conduction velocity and muscle weight. When measurements of multiple locations per nerve were reported, the most distal segment of the graft was used. In case the SEM was reported it was converted to SD (SD = SEM x √n). When outcome measure data was missing, authors were contacted for additional information. When data were displayed only graphically, we used Universal Desktop Ruler software (https://avpsoft.com/products/udruler/), to determine an adequate estimation of the outcome measurements. The mean of two independent measurements was used.

Statistical analysis

Data were analyzed using Review Manager, Version 5.4. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration. Meta-analysis was performed for all four outcome measurements by calculating the standardized mean difference (SMD) between vascularized and conventional grafts. Whenever a comparison reported an SD of 0 it was excluded from meta-analysis. A random effects model was applied, taking into account the accuracy of independent studies and the variation among studies and weighing all studies accordingly. Heterogeneity was measured using I2. Subgroup analyses were performed for different species (rabbit and rat), different graft length (0–2 cm, 2–4 cm and 4 > cm) and different time frames (0–2 months, 2–4 months and 4 > months). The results of subgroup analysis were only interpreted when groups consisted of 3 or more individual studies.

Funnel plots, egger regression and Trim and Fill analysis were used to search for evidence for publication bias if at least 10 or more studies per outcome. Because SMDs may cause funnel plot distortion, we plotted the SMD against a sample size-based precision estimate(1/√(n)).

To assess the robustness of our findings, a sensitivity analysis was performed. We evaluated the impact of excluding studies which used animals as their own control group.

Results

Study selection process

The search strategy presented in S1 Table retrieved 303 records, including 131 in PubMed and 172 in Embase. After removing duplicates, 203 articles appeared to be unique (Fig 1 shows a consort flow chart). After title abstract screening, 28 studies entered the full text screening phase. Finally, 14 articles were included in the review.

Fig 1

Flow chart of the study selection.

Study quality and risk of bias

This review clearly revealed that methodological details of animal experiments were often poorly reported. Reporting about any randomization and blinding measures taken in the conducted studies was respectively 21% (3 out of 14 publications).

The general results of our risk of bias assessment of the included references in this review are presented in Fig 2. Poor reporting of essential methodological details in most animal experiments resulted in an unclear risk of bias in the majority of studies. Risk of bias was scored separately for the 3 studies that used animals as their own control group because some aspects were not applicable (Fig 3).

Fig 2

Results of the risk of bias assessment of 11 included studies in this systematic review.

The first two items assess study quality by scoring reporting, a “yes” score indicates reported and a “no” score indicates unreported. The other items assessed risk of bias, with “yes” indicating low risk of bias, “no” high risk of bias, and “?” unclear risk of bias.

Fig 3

Results of the risk of bias assessment of the 3 included studies in this systematic review where animals were their own control group.

The first two items assess study quality by scoring reporting, a “yes” score indicates reported and a “no” score indicates unreported. The other items assessed risk of bias, with “yes” indicating low risk of bias, “no” high risk of bias, and “?” unclear risk of bias.

Study characteristics

The characteristics of the 14 included publications are shown in Table 1 [21-34]. All studies used either a rabbit (57%) or rat (43%) model. Notably, more than half the studies did not report gender (8 out of 14 studies). Out of the remaining studies 3 used females, 2 used males and in one both females and males were used. The sciatic nerve was the most commonly used nerve (50%), followed by the median nerve (29%), facial nerve (7%), peroneal nerve (7%) and auricular nerve (7%).

Table 1

The characteristics of all 14 included references.

Reference	Outcome measurements	Species	Gender	Animals	Grafts	Nerve	Muscle	Graft size (mm)	Time points (days)
Bertelli et al., 1996	Muscle weight	Rat	Female	70	70	Median	FCR	20	95, 120, 150, 210, 360
Donzelli et al., 2016	Axonal count	Rabbit	Male	20	20	Sciatic			30, 90
	Diameter
Hems et al., 1992	Axonal count	Rabbit	NR	8	8	Peroneal		50	250
Kanaya et al., 1992	Axonal count	Rat	Female	22	22	Sciatic	Tibialis anterior	25	84
	Nerve conduction velocity
	Muscle weight
Kawai et al., 1990	Axonal count	Rabbit	NR	34	67	Median		20,40, 60	56, 168
	Diameter
Koshima et al., 1985	Axonal count	Rat	Male	38	38	Sciatic		15	28, 56, 84, 112, 140, 168
	Diameter
Koshima.2 et al., 1985	Axonal count	Rat	NR	74	74	Sciatic		15	21, 28, 35, 42, 49, 56, 84,112, 140, 168, 224
	Diameter
Mani et al., 1992	Diameter	Rabbit	Male/Female	11	11	Sciatic		30	308
Matsumine et al., 2013	Axonal count	Rat	NR	14	14	Median		7	210
	Diameter
Ozcan et al., 1993	Axonal count	Rabbit	Female	10	10	facial		10	84
	Diameter
Seckel et al., 1986	Axonal count	Rat	NR	13	26	Sciatic		10	21, 28, 42
Shibata et al., 1988	Axonal count	Rabbit	NR	39	39	Median		30	70, 168
	Diameter
	Nerve conduction velocity
Tark et al., 2001	Axonal count	Rabbit	NR	33	66	Sciatic		40	56, 84, 112
Zhu et al., 2015	Nerve conduction velocity	Rabbit	NR	6	6	Auricular		20	112

NR: not reported.

FCR: flexor carpi radialis.

Axonal count

Data on axonal count could be retrieved from 11 independent studies containing 37 comparisons [22–27, 29–33]. Seven comparisons had to be excluded because not all outcome data was available, for example the SD. Out of the remaining 30 experiments conducted, data obtained from rabbits and rats was both 50%. In total 352 grafts were placed in 309 animals.

There was a variation in graft length from 7 to 60 mm. The graft length was unreported in two of the comparisons. Data were extracted at different time points varying between 21 and 250 days. The way axon count was measured differed. 27 out of the 30 comparisons used a field of interest of the nerve to determine the mean axonal count compared to three using the complete diameter. Generally, it was not mentioned how this field was chosen. Next to that, different formulas and computer programs were used to calculate the number of axons, such as a VAX or Compaq computer.

Overall analysis showed a significant difference in favor of treatment with a vascularized nerve graft (SMD, 0.46 [95% CI 0.06 to 0.86], N = 30) (Fig 4). The overall between study heterogeneity was moderate to high at I2 = 61%.

Fig 4

Forest plot of the effect of treatment with a vascularized nerve graft on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

Subgroup analyses revealed no differences in graft length, species and time frames when comparing axonal count between vascularized and conventional nerve autografts. The graft length middle group consisted of too few studies for subgroup analyses. (S1–S3 Figs).

Diameter

Eight studies, containing 31 comparisons, reported nerve fiber diameter on histological examination [22, 25–30, 32]. Since not all data was available, 10 of the 31 comparisons had to be excluded. Rabbits and rats were used in 52% and 48% respectively. All 21 comparisons combined, a total of 148 animals were operated on, resulting in 185 grafts that met our selection criteria. Graft length varied between 7 and 60 mm. In one of the studies, it was unclear which graft length was used. The time points at which data were extracted ranged from 21 to 308 days.

Analysis of all 21 included comparisons showed a significantly larger diameter after treatment with a vascularized nerve graft (SMD, 0.59 [95% CI 0.16 to 1.02], N = 21) (Fig 5). Study heterogeneity was moderate (I2 = 36%).

Fig 5

Forest plot of the effect of treatment with a vascularized nerve graft on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

The subgroup analysis for graft length could not be interpreted because both the middle and long group consisted of fewer than 3 studies.

For species however, there was a significant difference in diameter comparing rabbits and rats showing a more positive result in rats (SMD 0.13 [95% CI -0.28 to 0.54], N = 11; I2 = 11% compared to SMD 1.40 [95% CI 0.74 to 2.06], N = 10; I2 = 3%; P = 0.005). Rats showed a significant larger nerve fiber diameter in vascularized grafts compared to conventional grafts (S4 Fig).

A significant difference in diameter could not be found comparing different time frames (S5 Fig).

Nerve conduction velocity

Data on nerve conduction velocity could be extracted from 3 studies containing 4 comparisons [24, 32, 34]. Three comparisons used a rabbit model. A total of 74 animals were operated on, resulting in 74 grafts that met our selection criteria. Graft length ranged from 20 to 30 mm. Outcomes were measured at time points between 70 and 168 days.

Overall, analysis showed treatment with a vascularized nerve graft resulted in a significantly higher nerve conduction velocity (SMD, 1.19 [95% CI 0.19 to 2.19], N = 4) (Fig 6). Between studies, heterogeneity was high (I2 = 79%). There were not enough studies to perform a subgroup analysis.

Fig 6

Forest plot of the effect of treatment with a vascularized nerve graft on nerve conduction velocity.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

Muscle weight

Two studies, containing 6 comparisons, assessed muscle weight [21, 24]. A total of 92 animals, all rats, were operated on, resulting in 92 grafts. The two graft lengths used were 20 and 25mm. The varying time points at which data were extracted were between 84 and 360 days.

Overall, no significant difference was found between the treatment groups (SMD, 0.18 [95% CI -0,24 to 0,60], N = 6), I2 was 0% (Fig 7). There were not enough studies to perform a subgroup analysis.

Fig 7

Forest plot of the effect of treatment with a vascularized nerve graft on muscle weight.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

Sensitivity analyses

Axonal count

Exclusion of the studies in which animals were their own control group altered our results significantly. The previous effect in favor of a vascularized nerve graft compared to a conventional nerve autograft was no longer available (SMD 0.26 [95% CL -0.09 to 0.62], N = 18), heterogeneity was I2 = 17% (Fig 8). Conclusions of all subgroup analyses appeared to be robust (S6–S8 Figs).

Fig 8

Sensitivity analysis, forest plot of the effect of treatment with a vascularized nerve graft on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

Diameter

Exclusion of the studies in which animals were their own control group did not alter our results significantly. A significant difference in favor of a vascularized nerve graft compared to a conventional nerve autograft was found (SMD 1.03 [95% CL 0.39 to 1.68], N = 15), heterogeneity was I2 = 46% (Fig 9).

Fig 9

Sensitivity analysis, forest plot of the effect of treatment with a vascularized nerve graft on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL).

Next to that, the result of the subgroup analysis on species was altered. No significant difference in favor of rats was found. (SEM 0.39 [95% CI -0.68 to 1.45], N = 5; I2 = 62% compared to SEM 1.40 [95% CI 0.74 to 2.06], N = 10; I2 = 3%; P = 0.13) (S9 Fig). Other conclusions appeared to be robust (S10 Fig).

Publication bias analysis

Publication bias was assessed for axonal count only, because all other outcomes contained fewer than 10 studies.

Axonaffl count

The funnel plot suggested some asymmetry. Duval and Tweedie’s Trim and Fill analysis resulted in 6 extra data points (see Fig 10), indicating the presence of publication bias and some overestimation of the identified summary effect size.

Fig 10

Publication bias.

Discussion

This review suggests that a vascularized nerve graft does result in a significantly better nerve recovery compared to non-vascularized nerve autografts in animal models regarding the outcome measurements nerve fiber diameter, nerve conduction velocity and axonal count. However, the effect on axonal count did not appear to be very robust as after sensitivity analysis the effect was no longer present. Muscle weight did not differ between vascularized and non-vascularized grafts. Subgroup analysis indicated that the effect of vascularized graft on nerve fiber diameter is larger in rats compared to rabbits. However, this difference disappeared after sensitivity analysis.

A superior axon count and diameter was to be expected. A vascularized graft is believed to reduce intraneural fibrosis secondary to ischemia, which eases axonal regeneration. We expected a superior muscle weight as well because of a faster reinnervation reducing denervation of muscle atrophy. We think the small number of studies, that reported on muscle weight and therefore could be used for meta-analysis, is the reason for a lacking effect.

There is a lot of discussion on what the best outcome measurement for nerve regeneration is. Until this day there is no proper “gold standard” to test nerve recovery, although the ultimate goal of nerve recovery is to maximize sensation and motion. The most commonly used outcome measurement for sensation is the von Frey test [35]. For motion, walking track analysis was believed to be the best overall assessment [36-39]. At the moment it is rarely used and some would say it is even obsolete. Additionally, walking track analysis does not reflect maximum muscle force capacity. Others say the most precise measurement is the isometric response of muscle to tetanic contraction [40]. The authors are aware of the fact that histomorphometry, electrophysiology and axonal count in particular may have a limited correlated to the real functional recovery of sensation or motion [41]. However, despite the fact that these functional outcomes have the greatest clinical relevance, no data on functional recovery are present in the current literature.

This present meta-analysis of animal studies is, to the best of our knowledge, the first of its kind. Only some human case reports exist to try to put our findings into a broader perspective [8, 11, 42]. The clinical observations in these human case reports did not include the outcome measurements of this review. Nevertheless, all showed a superior sensory recovery in vascularized nerve grafts compared to conventional nerve grafts using different outcome measurements, such as the presence of a sharp/blunt discrimination, cold intolerance, the Tinel’s sign and the Semmes-Weinstein monofilament test.

Notably, clinical case reports found that vascularized nerve grafts give a better recovery in large nerve grafts compared to conventional nerve autografts. Terzis et al. [43] showed that a vascularized nerve graft successfully bridges a nerve defect longer than 13 cm where conventional nerve grafts generally fail. Also Xu et al. [44] and Okinaga et al. [45] concluded that when the graft length was short, the results were not significantly in favor of a vascularized nerve graft. However, we did not find a difference in recovery between various graft lengths in this meta-analysis.

As previously discussed a more significant donor site morbidity is one of the concerns surrounding a vascularized nerve graft. Donor site morbidity mainly depends on the harvested nerve, and the extend of the needed dissection. Therefore, we think it should also be assessed per case if the advantages of a vascularized graft justify the disadvantages of longer operative time and an enlarged donor site.

Limitations of this review

Firstly, our risk of bias analysis showed that most studies reported poorly on important methodological details. Therefore, most of the risk of bias items assessed had to be scored as unclear risk of bias. Even though this is quite commonly seen in animal studies, it is something to be taken into account [46]. The absence of reporting such methodological details could, to a certain extent, indicate the negligence of using these methods to minimize bias and confounding [47]. This can seriously hamper the possibility to draw reliable conclusions from the included animal studies.

Secondly, the number of studies included in this meta-analysis is relatively low, especially on nerve conduction velocity and muscle weight. This resulted in subgroups being relatively small, even to the extent that some subgroup analysis could not be interpreted. Furthermore, heterogeneity was moderate to high. However, because of their explorative nature a moderate to high heterogeneity between animal studies is expected.

To account for anticipated heterogeneity, we used a random effects model, conducted sensitivity analyses and explored the suggested causes for between study heterogeneity by means of subgroup analyses. Exploring this heterogeneity is one of the added values of meta-analyses of animal studies and might help to inform the design of future animal studies and subsequent clinical trials.

Thirdly, the graft length used to repair a nerve defect in rat and rabbit models is presumably smaller than those needed in humans. Therefore, the results shown in these animal experiments might not be correlated with the expected clinical outcomes.

Fourthly, a possible reason for heterogeneity could be the use of animals as their own control in some studies. Therefore, a sensitivity analysis was performed. This led to 3 studies being excluded because animals were used as their own control group. When Kawai et al. [25], Seckel et al. [31] and Tark et al. [33] were excluded there was not a significant difference in axonal count in favor of vascularized nerve grafts compared to conventional nerve autografts.

Fifthly, histomorphometry is difficult to compare between different laboratories, because other methods to measure the outcome were used. To compensate for these differences, we used a standardized mean difference for our meta-analysis. Over the years methods have evolved from manually calculating axonal count from a light microscopic photograph to a computer calculated estimate. The methods used by the studies in this review vary as well. Searching the publication databases, we found little evidence on which one is the best or on a clear sensitivity or specificity for these methods. Kim et al. [48] concluded that semi-automated method for counting axons in transmission electron microscopic images were strongly correlated with those of conventional counting methods and showed excellent reproducibility. Nevertheless, the techniques for histomorphometry will always be an estimation and therefore prone to bias.

Lastly, the presence of publication bias was identified. Our funnel plot suggested some asymmetry and Duval and Tweedie’s Trim and Fill analysis predicts some overestimation of the identified summary effect size of axonal count.

Conclusion

Treating a nerve gap with a vascularized graft results in superior nerve recovery compared to non-vascularized autografts nerve grafts in three out of four outcome measurements. However, this conclusion needs to be taken with some caution due to the inherent limitations of this meta-analysis. In addition, we recommend future studies to be performed under conditions more closely resembling human circumstances and to use long nerve grafts. Furthermore, we underline that future studies should use the Gold Standard Publication Checklist or ARRIVE guidelines to improve the reporting and methodological quality of animal studies [49, 50]. This is essential to improve the quality of the evidence presented in animal studies and the successful translation to humans in a clinical setting.

Subgroup analysis by graft length on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Short group vs. long group P = 0.07.

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Subgroup analysis by species on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Rabbit group vs. rat group P = 0.06.

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Subgroup analysis by time frames on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Short group vs. middle group P = 0.81. short group vs. long group P = 0.45.

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Subgroup analysis by species on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Rabbit group vs. rat group P = 0.005.

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Subgroup analysis by time frames on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Middle group vs. short group P = 0.12. Middle group vs. long group P = 0.08.

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Sensitivity analysis subgroup analysis by graft length on axonal count.

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Sensitivity analysis subgroup analysis by species on axonal count.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Rabbit group vs. rat group P = 0.62.

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Sensitivity analysis subgroup analysis by time frames on axonal count.

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Sensitivity analysis subgroup analysis by species on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Rabbit group vs. rat group P = 0.13.

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Sensitivity analysis subgroup analysis by time frames on diameter.

Data are presented as standardized mean difference (SMD) and 95% confidence intervals (CL). Middle group vs. long group P = 0.24.

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Raw data.

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Search strategy.

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23 Jun 2021

PONE-D-21-15417

Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis

This manuscript is a metanalysis comparing vascularized vs. no vascularized nerve graft. The article fits the scope of the journal. The title appropriately summarizes the article contents. The article is well-structured, the language is adequate. The references are uniform and according to the instructions for authors. The main concern is the heterogenous data with poor methodological details from the different manuscripts this paper is based on.

Please consider the next suggestions/clarification to improve the manuscript:

- Axonal account and diameter: were a uniform histological technique regarding the axonal account? Paper’s years of publication range from 1988 until 2016. Although these histological techniques are not novel, the sensitivity and specific of this technique has been improved during the last 30 years. Do the authors think it could exist a bias comparing these studies?

- I have a similar concern regarding the three studies with neurophysiology. This technique needs an experience neurologist or neurophysiologist to get reliable data.

I recommend the publication of the manuscript after the above comments are addressed. We thanks the authors the great effort needed for this study.

Reviewer #2: Thank you for your work and efforts to do a meta analysis.

What is the personal guidelines in your institution?

Please add your guideline into this paper.

The figures are in poor resolution, blurred and not clear.

Kindly ask you improve the resolution.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Aug 2021

Rebuttal letter

PONE-D-21-15417

“Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis”

Berend O. Broeren1*, Liron S. Duraku2, Caroline A. Hundepool2, Erik T. Walbeehm1, J. Michiel Zuidam2, Carlijn R. Hooijmans3,4, Tim De Jong1

PLOS ONE

Leila Harhaus

Academic Editor

PLOS ONE

Dear Ms Harhaus,

We thank you and the reviewers for a careful reading and the constructive comments regarding our manuscript and for the opportunity to revise and resubmit. We have addressed all recommendations and suggestions to further improve the manuscript. On behalf of my co-authors, I thank you for considering this revised manuscript for publication. We appreciate your time and look forward to your response.

Yours sincerely,

Berend Broeren (corresponding author)

berend.broeren@xs4all.nl

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis

This manuscript is a metanalysis comparing vascularized vs. no vascularized nerve graft. The article fits the scope of the journal. The title appropriately summarizes the article contents. The article is well-structured, the language is adequate. The references are uniform and according to the instructions for authors. The main concern is the heterogenous data with poor methodological details from the different manuscripts this paper is based on.

Please consider the next suggestions/clarification to improve the manuscript:

- Axonal account and diameter: were a uniform histological technique regarding the axonal account? Paper’s years of publication range from 1988 until 2016. Although these histological techniques are not novel, the sensitivity and specific of this technique has been improved during the last 30 years. Do the authors think it could exist a bias comparing these studies?

We appreciate the positive comments of Reviewer #1 and share the concerns regarding the heterogenous data and the poor methodological details from the different manuscripts this paper is based on. Therefore, it was extensively described in the section ‘limitations of this study” and the recommendation to use the Gold Standard Publication Checklist or ARRIVE guidelines for future animal studies to improve the reporting and methodological quality of animal studies in our conclusion was made. As to the uniformity of histological technique to measure axonal count, we added a section in the “limitations of this study” section. Different techniques were used by the studies in this review. To compensate for these differences, we used a standardized mean difference for our meta-analysis. The statement made by Reviewer #1 that these techniques have evolved is true and the sensitivity and specific of this technique may have been improved during the last 30 years. Over the years methods have evolved from manually calculating axonal count from a light microscopic photograph to a computer calculated estimate. Searching the publication databases, we found little evidence on which one is the best or on a clear sensitivity or specificity for these methods. Kim et al. concluded that semi-automated method for counting axons in transmission electron microscopic images were strongly correlated with those of conventional counting methods and showed excellent reproducibility. Nevertheless, the techniques for histomorphometry will always be an estimation and therefore prone to bias.

- I have a similar concern regarding the three studies with neurophysiology. This technique needs an experience neurologist or neurophysiologist to get reliable data.

The studies where neurophysiology was one of the outcome measurements did not mention the skillset of the person who assessed the outcomes. The data could possibly be biased but this cannot be established with the information at hand.

I recommend the publication of the manuscript after the above comments are addressed. We thanks the authors the great effort needed for this study.

Reviewer #2:

What is the personal guidelines in your institution? Please add your guideline into this paper.

Although we don’t have a clinical guideline for peripheral nerve gaps, we have added information of the clinical practice of our institution in the introduction of the manuscript.

The use of vascularized nerve grafts is debated in our medical centers because of the lack of clinical evidence indicating the superiority of a vascularized nerve graft, the concern of a more significant donor site morbidity compared to conventional nerve autografts and the complexity of the surgery.

The figures are in poor resolution, blurred and not clear.

Kindly ask you improve the resolution.

All figures have been improved using the PACE digital diagnostic tool to make sure the resolution improved and the PLOS requirements were met.

Submitted filename: Respons to Reviewers .docx

Click here for additional data file.

22 Oct 2021

PONE-D-21-15417R1Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysisPLOS ONE

Dear Dr. Broeren,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 06 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Leila Harhaus

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The study provided an interesting overview of the field of vascularized nerve grafts, indicating that vascularized nerve grafts can achieve superior results in terms of axon count, axon diameter, and nerve conduction velocity when compared to non-vascularized nerve grafts.

However, the manuscript needs some major and minor revision before publication.

Major Points

The introduction should include a more detailed explanation of the technique and rationale behind these techniques. The author discussed the disadvantages of the vascularized nerve graft but did not discuss its advantages over the conventional ANT.

Interestingly, the author emphasizes the critical nature of functional recovery but includes no data in the manuscript, implying that vascularized nerve grafts may have an effect on functional recovery. While we are well aware of the heterogeneity associated with assessing functional recovery following peripheral nerve injury, these issues should also be discussed in the manuscript.

This manuscript provided compelling evidence that a peripheral nerve surgeon should place a greater emphasis on the vascularized nerve graft. The author, however, did not discuss the findings under "Discussion."

What is the author's opinion about the superior axon count and diameter? Why do you see the effects even for the nerve conduction velocity and not for the muscle weight?

Justifies the advantages of an enlarged donor site the disadvantages?

Can the findings of an animal study be applied to humans?

Therefore the Discussion section should be appropriately revised.

Minor Points

Abstract

Line 54: What does this sentence mean? Three out of four measurements. In which measurements is a vascularized nerve graft superior?

Introduction:

The author should give a short overview of the technique of the vascularized nerve graft and explain its rationality further -

Line 63: When is it necessary to bridge the gap – when a tensionless coaptation is not possible – this should be added

Line 67: please rephrase the sentence – the meaning is clear – but the expression has to be improved

Line 80: what did these studies show – please elaborate on the findings of the previous research

Line 88: why do you use them and not nerve tubes? When you mean Axogen, then due to the results of the ranger study – these should be added.

Line 117: what about functional regeneration? Did the studies give no information focusing on the functional regeneration compared to a "conventional" ANT

Material and Methods:

Figure 1: Can you please clarify why you exclude the 175 manuscripts to classify the arguments. Would you please adjust the figure?

Results:

Line 205; the meaning of the sentence is not clear – please rephrase

Line 210: please add the level of significance

Line 235: why here SEM and not SMD?

Discussion:

This is a critical point in line 319 – 329. However, no data on functional recovery were provided, even with a small number of studies – despite the fact that this is the most important "test" for peripheral nerve injury recovery due to its clinical relevance.

Line 371: Because histomorphometry is crucial to this review, the author should explain how the axonal count was obtained. Counting axons using random field of interest methods and counting the entire diameter have varying degrees of validity for the data.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Nov 2021

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The study provided an interesting overview of the field of vascularized nerve grafts, indicating that vascularized nerve grafts can achieve superior results in terms of axon count, axon diameter, and nerve conduction velocity when compared to non-vascularized nerve grafts.

However, the manuscript needs some major and minor revision before publication.

Major Points

The introduction should include a more detailed explanation of the technique and rationale behind these techniques. The author discussed the disadvantages of the vascularized nerve graft but did not discuss its advantages over the conventional ANT.

We have added information on the technique and rationale behind the technique in the introduction of the manuscript. Page 4 line 72-74 and 76-80

Interestingly, the author emphasizes the critical nature of functional recovery but includes no data in the manuscript, implying that vascularized nerve grafts may have an effect on functional recovery. While we are well aware of the heterogeneity associated with assessing functional recovery following peripheral nerve injury, these issues should also be discussed in the manuscript.

We have added information on the rationale behind the choice of our outcome measurements and discuss the relevance of functional outcomes. Page 16 line 350, 351

This manuscript provided compelling evidence that a peripheral nerve surgeon should place a greater emphasis on the vascularized nerve graft. The author, however, did not discuss the findings under "Discussion."

The findings were indeed discussed in the “Discussion”.

This review suggests that a vascularized nerve graft does result in a significantly better nerve recovery compared to non-vascularized nerve autografts in animal models regarding the outcome measurements nerve fiber diameter, nerve conduction velocity and axonal count. Page 15 line 326-328

Treating a nerve gap with a vascularized graft results in superior nerve recovery compared to non-vascularized autografts nerve grafts in three out of four outcome measurements. Page 19 line 414, 415

What is the author's opinion about the superior axon count and diameter? Why do you see the effects even for the nerve conduction velocity and not for the muscle weight?

We have added information on how the authors think about the superior axon count and diameter and the lacking effect in muscle weight. See page 16 line 334-339

Justifies the advantages of an enlarged donor site the disadvantages?

We have added information on how the authors think about the question if the advantages of a vascularized nerve graft justify the disadvantages of an enlarged donor site. Page 17 line 366-370

Can the findings of an animal study be applied to humans?

We think this is a fair question. However, we also think this cannot be said with certainty. Animal research comes closed to clinical research and is therefore the most appropriate alternative if clinical research is not available. Of course, something can be said for the fact that research in primates comes closer to humans than, say, the rat. In particular when it comes to nerve grafts because of the length and diameter of the nerve graft. Therefore, we recommended future studies to be performed under conditions more closely resembling human circumstances and to use long nerve grafts.

Therefore the Discussion section should be appropriately revised.

Minor Points

Abstract

Line 54: What does this sentence mean? Three out of four measurements. In which measurements is a vascularized nerve graft superior?

This sentence was changed to clarify it. See page 3 line 54, 55

Introduction:

The author should give a short overview of the technique of the vascularized nerve graft and explain its rationality further –

Changed, page 4 line 72-74 and 76-80

Line 63: When is it necessary to bridge the gap – when a tensionless coaptation is not possible – this should be added

This had been added see page 3 line 64, 65

Line 67: please rephrase the sentence – the meaning is clear – but the expression has to be improved

This sentence has been rephrased see page 3 line 66-69

Line 80: what did these studies show – please elaborate on the findings of the previous research

Information has been added see page 4 line 82, 83

Line 88: why do you use them and not nerve tubes? When you mean Axogen, then due to the results of the ranger study – these should be added.

Did you mean line 98? Why we use autografts in most cases and not allografts? Autografts give a better outcome, with the down side of donor site morbidity which is minimal for most used nerve grafts. If not enough length of autografts is present, we use axogen.

We added references. See page 5 line 98, 99

Line 117: what about functional regeneration? Did the studies give no information focusing on the functional regeneration compared to a "conventional" ANT

No functional outcomes were described in more than one of the included articles. Only one described grasping strength and another one the sciatic function index.

Material and Methods:

Figure 1: Can you please clarify why you exclude the 175 manuscripts to classify the arguments. Would you please adjust the figure?

Figure was adjusted accordingly.

Results:

Line 205; the meaning of the sentence is not clear – please rephrase

Rephrased see page 10 line 215

Line 210: please add the level of significance

The significance was added, see page 11 line 226

Line 235: why here SEM and not SMD?

Changed to SMD, see page 12 line 250, 251

Discussion:

This is a critical point in line 319 – 329. However, no data on functional recovery were provided, even with a small number of studies – despite the fact that this is the most important "test" for peripheral nerve injury recovery due to its clinical relevance.

We rephrased this sentence. See page 16 line 350, 351

Line 371: Because histomorphometry is crucial to this review, the author should explain how the axonal count was obtained. Counting axons using random field of interest methods and counting the entire diameter have varying degrees of validity for the data.

We have added information on how axonal count was obtained in the Results of the manuscript. See page 11 line 220-224

Submitted filename: Respons to reviewers.docx

Click here for additional data file.

18 Nov 2021

Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis

PONE-D-21-15417R2

Dear Dr. Broeren,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Leila Harhaus

Academic Editor

PLOS ONE

22 Nov 2021

PONE-D-21-15417R2

Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: a systematic review and meta-analysis

Dear Dr. Broeren:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. med. Leila Harhaus

Academic Editor

PLOS ONE