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Umbilical cord-derived Wharton’s Jelly for
Regenerative Medicine Applications in Orthopedic
Surgery: A Systematic Review Protocol
Benjamin J. Main
Beaumont Hospital - Farmington Hills
Josiah A. Valk
Beaumont Hospital - Farmington Hills
Nicola Maffulli
Barts and The London School of Medicine and Dentistry
Hugo C. Rodriguez
University of the Incarnate Word School of Osteopathic Medicine
Manu Gupta
Future Biologics
Ian W. Stone
University of the Incarnate Word School of Osteopathic Medicine
Saadiq F. El-Amin III
BioIntegrate
Ashim Gupta ( ashim6786@gmail.com )
Future Biologics https://orcid.org/0000-0003-1224-2755
Study protocol
Keywords: Regenerative Medicine, Musculoskeletal Injuries, Umbilical Cord, Wharton’s Jelly, PRISMA
DOI: https://doi.org/10.21203/rs.3.rs-34938/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Background: Musculoskeletal injuries and conditions affect millions of individuals.These ailments are
typically managed by immobilization, physiotherapy or activity modication. Regenerative medicine has
experienced tremendous growth in the past decades, especially in musculoskeletal medicine. Umbilical
cord-derived Wharton’s Jelly is an exciting new option for such therapies. Wharton’s jelly is a connective
tissue located within the umbilical cord largely composed of mesenchymal stem cells and extracellular
matrix components, including collagen, chondroitin sulfate, hyaluronic acid and sulfated proteoglycans.
Wharton’s Jelly is a promising and applicable biologic source for orthopedic regenerative application.
Methods: A systematic search will be conducted in PubMed, ScienceDirect and Google Scholar
databases of English, Italian, French, Spanish and Portuguese language articles published to date.
References will be screened and assessed for eligibility by two independent reviewers as per PRISMA
guidelines. Articles will be considered without exclusion to sex, activity or age. Studies will be included if
they used culture-expanded, mesenchymal stem/stromal cells of mesenchymal stem cells and/or
connective tissue obtained from Wharton’s Jelly. Studies will be excluded if Wharton’s Jelly is not the sole
experimental examined cell type. Placebos, conventional non-operative therapies including steroids
injections, exercise and NSAIDs will be compared. The study selection process will be performed
independently by two reviewers using a reference software. Data synthesis and meta-analysis will be
performed separately for clinical and pre-clinical studies.
Discussion: The results will be published in relevant peer-reviewed scientic journals. Investigators will
present results at national or international conferences.
Trial registration: The Protocol was registered on PROSPERO international prospective register of
systematic reviews prior to commencement, CRD42020182487.
Background
Musculoskeletal conditions affect millions of individuals each year and represent a large burden on
healthcare. These ailments are classically managed with immobilization, activity modication, physical
therapy, and pharmacological agents, and surgery when conservative management has failed. These
modalities are limited, often attempting to limit pain rather than addressing the actual pathology [1-7].
Regenerative medicine has undergone tremendous growth during the past few decades, especially in
musculoskeletal medicine [8]. Human stem cells offer regenerative potential, aiming to hasten or reverse
chronic disease in order to restore function [9-11]. Common sources of metabolically available human
stem cells include bone marrow concentrate (BMC), adipose tissue, amniotic tissue, umbilical cord blood
and umbilical cord-derived Wharton’s Jelly [12-17]. Stem cell biologic therapies for the treatment of
musculoskeletal injuries are becoming more common in orthopedic clinical practice. Increased patient
awareness has led to increased demand for biologic therapies [8].
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To date, there is no consensus regarding the most appropriate stem cell source. BMC, PRP, and adipose-
derived stem cells (ADSCs) have received increased scientic attention given their unique properties and
clinical availability. Direct injection therapy has proven benecial in a human model. Bone marrow
mesenchymal stem cells (BMSCs) have limitations associated with increased pain and morbidity from
the bone marrow aspiration procedure [18]. BMC has a limited number of BMSCs, with roughly (0.001-
0.01%) of BMSCs within the BMC [18]. The limited availability and the fact that BMSCs show signs of
early senescence urge to identify and exploit other sources of stem cells [18]. The literature on ADSCs is
limited, with the lack of randomized double-blinded trials and lack of long term follow up observation
being the major contributors [19]. The long-term safety of ADSCs is also yet to be determined [19]. PRP
has been extensively studied, but most of the results are biased, and from poorly designed studies that
favor the publication of positive results [20]. Although safe and promising, PRP still has not shown strong
evidence of ecacy and effectiveness in the human clinical setting [20].
Wharton’s Jelly is a connective tissue located within the umbilical cord largely composed of hyaluronic
acid and chondroitin sulfate. The chief role of Wharton’s Jelly is to resist torsional and compressive
stresses imposed upon the umbilical vessels during fetal development. Within the Wharton’s Jelly are
primitive mesenchymal stem cells [21]. These perinatal mesenchymal stem cells resemble embryonic
stem cells, but, retain many properties of adult mesenchymal stem cells. Wharton’s Jelly-derived
mesenchymal stem cells express lower levels of pluripotent markers than embryonic stem cells,
suggesting they are highly multipotent rather than pluripotent [22, 23]. Wharton’s Jelly contains the
highest concentration of mesenchymal stem cells per milliliter compared to other tissues with rich
extracellular matrix components, including collagen, chondroitin sulfate, hyaluronic acid, and sulfated
proteoglycans [24, 25]. Wharton’s Jelly has also a clinically relevant quantity of growth factors, cytokines
and extracellular vesicles [1]. The large amount of these substances may play a role in reducing
inammation, pain and augment healing of musculoskeletal injuries [1].
Wharton’s Jelly is easily accessible and available as opposed to other biologics. Every birth presents an
opportunity to harvest these highly multipotent, nutrient rich mesenchymal stem cells. The ease of
collection offers many advantages over current BMC and adipose-derived stem cell harvest, which pose
donor site morbidity. This factor, as well as the attractive expansion properties of umbilical-derived stem
cells and clinically signicant amounts of applicable regenerative substances,make Wharton’s Jelly a
promising source of mesenchymal stem cells for orthopedic regenerative application [1, 26]. Considering
the lack of clarity regarding the use of Wharton’s jelly, methodological factors, clinical translation and
outcome measurement, a systematic review is required to synthesize and evaluate the quality of the
available evidence regarding the safety and ecacy of Wharton’s jelly for regenerative medicine
applications in orthopedic surgery. The primary objective of this review is to report the clinical, structural
and functional outcomes of the applications of Wharton’s jelly for regenerative medicine in orthopedic
surgery. The secondary objective is to identify methodological characteristics associated with application
outcomes.
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Methods
The protocol was registered on the PROSPERO international prospective register of systematic reviews,
registration number CRD42020182487. The systematic review will follow the Preferred Reporting Items
for Systematic Reviews and Meta-Analyses (PRISMA) statement and guidelines [27, 28].
Eligibility Criteria
The PICOS (Population, Intervention, Comparison, Outcome, Study Design) framework will be used as a
template for dening eligibility criteria for literature search [29]. Characteristics for clinical studies are as
follow:
Population
Research involving animal and human models (clinical studies as well as
in vitro
research) will be
considered for review, without exclusion relating to sex or age. Basic science studies must include human
cells isolated from a living model. Studies meeting eligibility criteria will apply to acute orthopedic
musculoskeletal injury, chronic orthopedic musculoskeletal conditions, or an articial disease model.
Articles will be excluded if they do not relate to orthopedic intervention.
Intervention
Research meeting inclusion criteria will involve the use of mesenchymal stem cells and/or connective
tissue obtained from Wharton’s Jelly. Studies will be excluded if Wharton’s Jelly is not the experimental
examined cell type. Studies involving umbilical derived stem cells will be excluded unless the study
species the use of Wharton’s Jelly. Studies will be excluded if they report the use of Wharton’s Jelly
derived mesenchymal stem cells in combination with other cell populations.
Comparison
Comparators considered will include placebos, non-injury models, acute injury models, non-injury models,
and gold standard treatments for orthopedic injury.
Outcomes
For basic scientic research, studies relating to human musculoskeletal injury via histological or
biochemical measures will be included. For clinical research, studies pertaining to human or animal
orthopedic musculoskeletal injury via histological and/or biochemical measures and functional scores
(pain, activity, quality of life, etc.) will be included.
Study Design
Observational studies (cohort, cross-sectional and case-controlled prospective or retrospective studies) or
randomized controlled trials comparing outcomes of connective tissue derived from Wharton’s Jelly with
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control, experimental therapy, or gold standard treatment at any follow up period will be included.
Systematic reviews will only be examined to identify further studies for inclusion, and results of meta-
analysis will not be included in the analysis. With regard to publication year, all studies published to date
will be included.
Information Sources
A systematic search will be conducted in PubMed, ScienceDirect and Google Scholar databases of
English, Italian, French, Spanish and Portuguese language articles published before May 2020.
Secondary searching of reference lists of key articles and reviews will be undertaken to identify any
additional studies potentially missed in electronic search.
Search
The search and selection process will be based on the PRISMA checklist and ow diagram based on the
eligibility and inclusion criteria previously outlined. A web-based reference software system (RefWorks)
will be used for data management.
Study Selection
The study selection process will be performed independently by two reviewers. Screening of abstracts will
be performed, and full text articles will be retrieved and uploaded to the reference software. A thorough
secondary screening will be performed independently by two reviewers. The secondary screening of the
full text articles will eliminate studies that do not meet inclusion criteria.
Data Collection
Data extraction from articles that meet inclusion criteria will be performed by two independent reviewers.
Data extracted and synthesized will include authors, publication year, study design, group controls, group
interventions, outcome measurement, and outcome assessment. Customized forms will be used in the
data extraction and collection process. The primary authors will be contacted via email for any
information necessitating clarication.
Data Items
Relevant items of population, problem, intervention, comparison, and outcome will be extracted and
included. For basic scientic research, relevant histological and/or biochemical measures will be
included. For clinical research, all histological measures, biochemical measures and functional scores
will be included.
Risk of bias
Multiple tools will be used to assess the risk of bias for included studies. The Systematic Review Centre
for Laboratory Animal Experimentation (SYRCLE) risk of bias tool will be applied to animal studies [30].
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Ten domains will be addressed related to selection bias, performance bias, detection bias, attrition bias,
reporting bias, and other biases. The Risk Of Bias In Non-randomized Studies of Interventions (ROBINS-I)
tool will be used to assess observational and quasi-randomized studies [31]. Seven domains will be
utilized to assess risk including: confounding, participant selection bias, classication bias, deviation
bias, bias due to missing data, outcome measurement bias, and bias in selection of reported results.
Studies will be judged to have no information or a low, moderate, serious, or critical risk of bias. For
randomized control trials, the Risk of Bias 2 (RoB 2) tool will be used to establish risk of bias [32]. Five
domains including biases arising from the randomization process, due to deviations from intended
interventions, due to missing outcome data, in measurement of the outcome, and in selection of the
reported result will be analyzed. Overall risk of bias will be determined to be low, some concerns, or high.
All included studies will be independently scored by two reviewers, and consensus reached by discussion.
Data synthesis and meta-analysis
Data synthesis and meta-analysis will be performed separately for clinical and pre-clinical studies,
following the guidelines published by Hooijmans et al [30]. Given the sparsity of homogenous research, a
qualitative analysis of common outcome variables will be conducted. Subgroups chosen for analysis will
include cells derived from Wharton’s Jelly versus experimental therapy and/or control. Results of meta-
analyses extracted data will be summarized in tables and narrative interpretation provided, with
emphasis on outcome measures.
Discussion
The results of this review will be published in a relevant scientic journal or presented at national or
international conferences (‘Publications’) by the Investigators.
Documenting protocol amendments
Protocol amendments and updates will be documented via PROSPERO online register. The nature of the
changes made will be recorded, dated and accessible along with the most recent version within the record
audit trail under the systematic review protocol registration number CRD42020182487.
List Of Abbreviations
ADSCs Adipose-derived stem cells
BMC Bone marrow concentrate
BMSCs Bone marrow mesenchymal stem cells
PICOS Population, Intervention, Comparison and Outcome
PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses
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PRP Platelet-rich plasma
ROBINS-I Risk Of Bias in Non-randomized Studies- of Interventions
SYRCLE Systematic Review Centre for Laboratory Animal Experimentation
Declarations
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Availability of data and materials
The results of this review will be published in a relevant scientic journal or presented at national or
international conferences (‘Publications’) by the Investigators.
Competing interests
The authors declare that they have no competing interests.
Funding
This research received no specic grant from any funding agency in the public, commercial or not-for-
prot sectors.
Authors’ contributions
AG and NM contributed to the review concept and study design. BJM, JAV and AG provided input for the
review. BJM and JAV provided input to the development of search strategies and methodologies for the
literature review. BJM, JAV, HCR, MG, IWS, NM, SFE and AG drafted the review protocol. All authors
provided feedback and approved the nal protocol. AG is the guarantor of the review.
Acknowledgments
Not Applicable
References
1. Gupta A, El-Armin SF 3rd., Levy HJ, Sze-Tu R, Ibim SE, Maffulli N. Umbilical cord-derived Wharton’s
jelly for regenerative medicine applications. J Orthop Surg Res. 2020 Feb;15(1):49.
Page 8/9
2. Navani A, Manchikanti L, Albers SL, Latchaw RE, Sanapati J, Kaye AD, et al. Responsible, Safe, and
Effective Use of Biologics in Management of Low Back Pain: American Society of Interventional Pain
Physicians (ASIPP) Guidelines, et al. Pain Physician. 2019 Jan;22(1s):S1-S74.
3. Gupta A, Woods MD, Illingworth KD, Niemeier R, Schafer I, Cady C, et al. Single walled carbon
nanotube composites for bone tissue engineering. J Orthop Res. 2013 Sep; 31(9): 1374-1381.
4. Gupta A, Main BJ, Taylor BL, Gupta M, Whitworth CA, Cady C, et al. In vitro evaluation of three-
dimensional single-walled carbon nanotube composites for bone tissue engineering. J Biomed Mater
Res A. 2014 Nov;102(11):4118-26.
5. Gupta A, Liberati TA, Verhulst SJ, Main BJ, Roberts MH, Potty AG, et al. Biocompatibility of single-
walled carbon nanotube composites for bone regeneration. Bone Joint Res. 2015 May;4(5):70-7
6. Vallejo R, Gupta A, Kelly CA, Vallejo A, Rink J, Williams JM, et al. Effects of Phase Polarity and
Charge Balance Spinal Cord Stimulation on Behavior and Gene Expression in a Rat Model of
Neuropathic Pain. Neuromodulation. 2020 Jan:23(1):26-35.
7. Gupta A, Sharif K, Walters M, Woods MD, Potty A, Main BJ, et al. Surgical retrieval, isolation and in
vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications.
J Vis Exp. 2014 Apr 30;(86).
8. Lamplot JD, Rodeo SA, Brophy RH. A practical Guide for the Current Use of Biologic Therapies in
Sports Medicine. Am J Sports Med. 2020 Feb;48(2)488-503.
9. Caplan AI. Mesenchymal stem cells. J Ortho Res. 1991 Sep;9(5):641-50.
10. Minguell JJ, Erices A, Conget P. Exp Biol Med (Maywood). 2001 Jun;226(6):507-20.
11. Andia I, Maffulli N. New biotechnologies for musculoskeletal injuries. Surgeon. 2019 Aug;17(4):244-
255.
12. Le ADK, Enweze L, DeBaun MR, Dragoo JL. Current Clinical Recommendations for Use of Platelet-
Rich Plasma. Curr Rev Musculoskelet Med. 2018 Dec;11(4):624-634.
13. Patel JM, Saleh KS, Burdick JA, Mauck RL. Bioactive factors for cartilage repair and regenerations:
Improving delivery, retention, and activity. Acta Biomater. 2019 Jul 15;93:222-238.
14. Le ADK, Enweze L, DeBaun MR, Dragoo JL. Platelet-rich Plasma. Clin Sports Med. 2019 Jan;38(1):17-
44.
15. Sezgin EA, Atik OS. Are orthobiologics the next chapter in clinical orthopedics? A literature review.
Eklem Hastalik Cerrahisi. 2018 Aug;29(2):110-6.
16. Duerr RA, Ackermann J, Gomoll AH. Amniotic-Derived Treatments and Formulations. Clin Sports Med.
2019 Jan;38(1):45-59.
17. Chirichella PS, Jpw S Iacono S, Wey HE, Malanga GA. Treatment of Knee Meniscus Pathology:
Rehabilitation, Surgery, and Orthobiologics. PM R. 2019 Mar;11(3):292-308.
18. Mohamed-Ahmed S, Fristad I, Lie SA, Suliman S, Mustafa K, Vindenes H, et al. Adipose-derived and
bone marrow mesenchymal stem cells: a donor-match comparison. Stem Cell Res Ther. 2019;168(9).
Page 9/9
19. Usuelli FG, D’Ambrosi R, Maccario C, Indino C, Manzi L, Maffulli N. Adipose-derived stem cells in
orthopaedic pathologies. Br Med Bull. 2017 Dec1;124(1):31-54.
20. Brossi PM, Moreira JJ, Machado TS, Baccarin RY. Platelet-rich plasma in orthopedic therapy: a
comparative systematic review of clinical and experimental data in equine and human
musculoskeletal lesions. BMC Vet Res. 2015 Apr22;11:98.
21. Troyer DL, Weiss ML. Concise review: Wharton’s jelly-derived cells are a primitive stromal cell
population. Stem Cells 2008;26(3):591e9.
22. Carlin R, Davis D, Weiss M, Schultz B, Troyer D. Expression of early transcription factors Oct-4, Sox-2
and Nanog by porcine umbilical cord (PUC) matrix cells. Reproductive Biology and Endocrinology
2006;4(1):8.
23. La Rocca G, Anzalone R, Corrao S, Magno F, Loria T, Lo Iacono M, et al. Isolation and characterization
of Oct-4þ/HLA-Gþ mesenchymal stem cells from human umbilical cord matrix: differentiation
potential and detection of new markers. Histochemistry and Cell Biology 2009;131(2):267e82.
24. Vangsness CT Jr, Sternberg H, Harris L. Umbilical cord tissue offers the greatest number of
harvestable Mesenchymal stem cells for research and clinical application: a literature review of
different harvest sites. Arthroscopy. 2015;31:1836–43.
25. Sobolewski K, Małkowski A, Bańkowski E, Jaworski S. Wharton’s jelly as a reservoir of peptide growth
factors. Placenta. 2005;26:747–52.
26. Schugar RC, Chirieleison SM, Wescoe KE, Schmidt BT, Askew Y, Nance JJ, et al. High harvest yield,
high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive
human umbilical cord tissue. Journal of Biomedicine and Biotechnology; 2009. Article ID 789526:11.
27. Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for
Systematic Reviews and Meta-Analyses: The PRISMA Statement. Open Med 2009; 3(3); 123-130.
28. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, et al. (2009) The PRISMA Statement for
Reporting Systematic Reviews and Meta-Analyses of Studies That Evaluate Health Care
Interventions: Explanation and Elaboration. BMJ 2009;339:b2700, doi: 10.1136/bmj.b2700
29. O’Connor D, Green S, Higgins J. Dening the review question and developing criteria for including
studies, Cochrane handbook for systematic reviews of interventions: Cochrane book series,
Chichester, West Sussex:Wiley;2008.p.81-94.ISBN: 978-0-470-51845-8.
30. Hooijmans CR, Rovers MM, de Vries RB, Leenaars M, Ritskes-Hoitinga M, Langendam MW. SYRCLE’s
risk of bias tool for animal studies. BMC Med Res Methodol. 2014 Mar 26; 14:43.
31. Sterne JA, Hernán MA, Reeves BC, Savović J, Berkman ND, Viswanathan M, et al. ROBINS-I: a tool for
assessing risk of bias in non-randomized studies of interventions. BMJ. 2016 Oct 12;355:i4919.
32. Sterne JAC, Savović J, Paige MJ, Elbers RG, Blencowe NS, Boutron I, et al. RoB 2 a Revised tool for
assessing risk of bias in randomised trials. BMJ. 2019 Aug 28;366:I4898.