A Systematic Review of Bone Marrow Stromal Cells and Periosteum-Derived Cells for Bone Regeneration

Abstract

Bone marrow stromal cells (BMSCs) and periosteum-derived cells (PDCs) represent promising skeletal stem cell sources to treat critical-size bone defects. However, the large number of preclinical tests with a variety of in vivo data complicates the selection of cells for further clinical translation. This systematic review aims to analyze the in vivo bone-forming efficacy of BMSCs- and PDCs-based approaches in all published preclinical experiments until November 2020. For this purpose, four databases (PubMed, Embase, Cochrane Central Register of Controlled Trial, and Web of Science) were searched for eligible literature, which yielded a total of 94 full-text articles for systematic review. This review generated an evidence-based list of BMSC- or PDC-based approaches, which have been evaluated for bone formation in different animal models. Among them, 74 studies were included for pairwise and network meta-analysis. The results revealed that both PDC and BMSC had beneficial bone-forming efficacy compared to bare scaffold. In addition, BMSC- and PDC-based approaches had no significant difference regarding in vivo bone-forming efficacy. However, BMSC-based approach had a higher probability to be ranked better than PDC-based approach. Furthermore, the review discusses (i) the possible risk of bias of the in vivo evaluation of cell-based approaches, (ii) the difficulty in replication of such experiments due to frequent poor reporting of the methods and results, and (iii) the clinical relevance of the currently utilized BMSC- and PDC-based approaches. Systematic review registration: The study was prospectively registered in PROSPERO, Registration No. CRD42021270922.

Impact statement

Bone marrow stromal cells (BMSCs) and periosteum-derived cells (PDCs) represent promising skeletal stem cell sources to treat critical-size bone defects. However, a large number of preclinical tests with a variety of in vivo data complicate the selection of cells for further clinical translation. This review goes beyond summarizing the existing experimental evidence of BMSCs and PDCs by systematically evaluating their performance in bone formation in different defect models. This evidence-based list of well-documented preclinical evaluations will serve as a practical guideline in future research on cell-based therapies for bone repair and reconstruction.

Introduction

Bone tissue possesses regenerative potential, however, bone defects that exceed critical sizes still cannot heal through the natural healing.¹ Hence, the reconstruction of large bone defects caused by trauma, congenital diseases, and tumor section has been a great clinical challenge.^2,3 Nowadays, the gold standard to treat complex large bone defects involves transplantation of autogenous or allogenous bone grafts, which can be harvested from the iliac crest, fibula, scapula, or radius.⁴ However, the inherent limitations of this approach, including insufficient autogenous resources and donor-site morbidity,⁵ strongly urge clinicians and researchers to explore alternative treatment options.

Cell-based approach arises as a promising strategy to treat complex and large bone defects, especially in compromised conditions. It is envisioned that the stem/progenitor cells with the ability to self-renew and differentiate are the key factors in steering bone formation and integration, subsequently leading to the successful healing of the defect.^6,7 Therefore, recent research has been focused on the development of cell-based approaches for bone regeneration to overcome the limitations of the existing treatments.

Among various cell types for bone regeneration, bone marrow aspirate and periosteum represent two reliable tissue resources to harvest skeletal stem/progenitor cells with convincing evidence of differentiation capacity both in vitro and in vivo.^8–13 Although the bone marrow stromal cells (BMSCs) and periosteum-derived cells (PDCs) shared similarities in trilineage differentiation capacity and surface markers,^9,14 they showed different in vitro and in vivo biological performance. For instance, the in vitro proliferation and differentiation capacity of BMSCs diminishes as aging,¹⁵ and after implantation in vivo, BMSCs have been shown to exclusively contribute to fracture healing by intramembranous ossification.¹⁶ On the contrary, PDCs showed a stable in vitro differentiation capacity after several passages, regardless of the donor age.¹⁷ Furthermore, PDCs contributed to the majority of cartilage callus in the defect site,^9,16 which is an essential process for the natural healing of bone fracture.¹⁸

Despite numerous studies in both preclinical and clinical settings,^{8,11,12,19–26} the efficacy of cell-based approaches for bone regeneration remains controversial. Some studies showed that BMSCs-based approaches had a favorable effect on bone formation compared to their controls (e.g., bare scaffold group when the bare scaffold was applied,^27–29 while other studies draw the opposite conclusions³⁰ or reported that there was no significant difference between experiments with or without BMSCs.^31,32 The conclusions regarding the efficacy of PDCs also varied. Some studies proved that PDC-based approaches had better efficacy at new bone formation than bare scaffold,^33,34 while others demonstrated no significant difference²⁰ or the opposite.²² Furthermore, different opinions exist regarding the choice of cell types. Zhu et al³⁵ reported that PDCs-based approaches showed a superior subcutaneous bone formation compared to BMSCs-based approaches, while other studies showed different conclusions.^{21,33,36–38}

To increase the value of animal experimental work as proofs-of-concept before entering new trials, it is worthwhile to perform a systematic analysis of all preclinical data to determine their potential translational value for human application. Consequently, the purpose of this work was to perform a systematic review and meta-analysis regarding BMSCs- or PDCs-based approach for bone regeneration to obtain more clarity on their in vivo bone-forming efficacy.

Materials and Methods

This review was designed and conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines for systematic review incorporating the network meta-analysis.³⁹ The protocol was registered in the PROSPERO database (CRD42021270922).

Search strategy

Electronic bibliographic databases of MEDLINE/PubMed, EMBASE, Cochrane Central Register of Controlled Trial, and Web of Science were searched from the inception of each database to November 2, 2020. Literature concerning PDC and BMSC was separately searched. The full search strategy was based on four search components: “periosteum-derived cell” (or “bone marrow stromal cell”), “bone regeneration,” “tissue engineering,” and “bone.” Detailed search strategies for PubMed are displayed in Table 1 (the adapted strategies for the other three databases can be found in Supplementary Table S1). No language restrictions were imposed. A manual search was also conducted by searching the major journals of the same field by hand. Bibliographies of selected studies and review articles on the same topic were checked for cross-reference. Unpublished data were searched on OpenGrey.

Table 1.

Search Strategy for PubMed

Component	Search	Query	Results
PDC-based studies	#7	#1 AND #2 AND #3 AND #5	553
BMSC-based studies	#6	#1 AND #2 AND #3 AND #4	3421
PDC	#5	((((periosteal cell) OR (periosteum derived cell)) OR (periosteum cell)) OR (periosteum osteoblast)) OR (periosteum derived osteoblast)	4696
BMSC	#4	((((bone marrow stem cell) OR (bone marrow stromal cell)) OR (bone marrow mesenchymal stem cell)) OR (bone marrow mesenchymal cell)) OR (bone marrow derived cell)	161,445
Bone regeneration	#3	(((((((((((((bone regeneration[Title/Abstract]) OR (bone repair[Title/Abstract])) OR (osteogenesis[Title/Abstract])) OR (osteogenic[Title/Abstract])) OR (ossification[Title/Abstract])) OR (bone formation[Title/Abstract])) OR (bone reconstruction[Title/Abstract])) OR (bone transplant[Title/Abstract])) OR (bone graft^*[Title/Abstract])) OR (bone augmentation[Title/Abstract])) OR (bone healing[Title/Abstract])) OR (bone engineering[Title/Abstract])) OR (bone tissue engineering[Title/Abstract])) OR (engineered bone[Title/Abstract])	130,439
Tissue engineering	#2	((((((((((((((stem cell[MeSH Terms]) OR (cell culture technique[MeSH Terms])) OR (cells, cultured[MeSH Terms])) OR (cell culture[MeSH Terms])) OR (cell transplantation[MeSH Terms])) OR (cell engineering[MeSH Terms])) OR (tissue engineering[MeSH Terms])) OR (tissue culture technique[MeSH Terms])) OR (tissue culture[MeSH Terms])) OR (regenerative medicine[MeSH Terms])) OR OR (tissue engineered[Title/Abstract])) OR (bone engineering[Title/Abstract])) OR (bioengineer[Title/Abstract])) OR (cell-based[All Fields])) OR (cell seeded[Title/Abstract])	1,955,746
Bone	#1	bone[MeSH Terms]	612,873

BMSC, bone marrow stromal cells; PDC, periosteum-derived cell.

Screening and selection of articles

Studies were included when they fulfilled all the following criteria: (1) Preclinical experiments that involved transplantation of BMSC- or PDC-based approaches; (2) Outcome data were presented as quantitative new bone formation (%) after the local application of BMSC- or PDC-based approaches; and (3) Full-text original article. Studies that fulfilled the following criteria were excluded: (1) Models with orthopedic disorders; (2) Coculture of different cells; and (3) Cells were genetically modified or not isolated from the original tissue.

The selection of eligible studies was done in duplicate by two independent reviewers (J.Z. and J.X.) based on the abovementioned inclusion and exclusion criteria. Initial screening was conducted on the titles and abstracts. Eligible studies based on the initial screening were reassessed in the full-text screening for eligibility for inclusion. Any disagreement during these two phases will be resolved through discussion or consulting the third reviewer (W.J.).

Study characteristics and data extraction

The following study characteristics were extracted: the number of animals/defects, animal species, animal sex, implant site, defect size, scaffold material, cell loading number, cell source, healing duration, first author, year of publication, and any other reported adverse events.

The new bone formation (%) measured through histomorphometry was quantified as the outcome measurements used for the meta-analysis. Due to the limited number of the included studies, the results measured by micro-computed tomography (CT) were not used for meta-analysis.

The mean of new bone formation percentage, standard deviation (SD) or standard error of the mean (SEM), and the number of defects per group (n) were recorded or recalculated. If the new bone formation is displayed graphically, data were extracted using ImageJ (1.53a, National Institutes of Health, USA) when possible.⁴⁰ If SEM was reported, the SEM was converted to SD. Independent experiments within one article were extracted separately.

Risk of bias assessment

The risk of bias of the included studies was assessed by two reviewers (J.Z. and J.X.) independently using an adapted version of SYRCLE's risk of bias tool.⁴¹ “1. Was the allocation sequence adequately generated and applied? 2. Were the groups similar at baseline or adjusted for confounders? 3. Was the allocation adequately concealed? 4. Were the animals randomly housed during the experiment? 5. Were the caregivers and/or investigators adequately blinded? 6. Were animals selected at random during outcome assessment? 7. Was the outcome assessment adequately blinded? 8. Were incomplete outcome data adequately addressed?” For each item, the risk of bias in the included studies was rated as “high,” “low,” or “unclear.” In item 4, the risk of bias was considered low if the animals were housed singly.

To avoid too many items being rated as unclear due to the poor reporting quality of animal studies, two more questions were added: “(a) Was it stated that the experiment was randomized at any level? and (b) Was it stated that the experiment was blinded at any level?”⁴²

Data synthesis and statistical analysis

First, a DerSimonian–Laird random-effect pairwise meta-analysis was performed on the outcome measures by computing the standardized mean difference (SMD). Data were analyzed with Stata 16 (StataCorp. 2019).⁴³ If one reference studied the bone-forming efficacy of two types of cell-based approaches or at various cell seeding densities in different groups of animals, these groups were analyzed as independent experiments. If one control group served more than one experimental group, the number of defects in the control group was divided by the number of experimental groups it served. When several time pointes were assessed, the data at the last time point were extracted.

Subgroup analysis was divided based on the animal model species. Then, the combined effect size (ES) and variance were calculated. Individual and weighted overall ES was displayed using a forest plot. Data were presented as Hedges'g and 95% confidence intervals (CIs). The heterogeneity in the estimates across the different studies was assessed using the Cochrane test for heterogeneity and the I² statistic. Potential publication bias was assessed by using Egger's linear regression test and funnel plots.

Next, a frequentist random-effect network meta-analysis was performed using the network commands package in Stata.^43,44 Inconsistency between the direct and indirect evidence obtained from network meta-analysis was assessed by using the global inconsistency evaluation and loop inconsistency model. Heterogeneity was represented with I² statistics. Odds ratio (OR) and 95% CI were calculated to compare the effect between groups. The surface under the cumulative ranking (SUCRA) probabilities was then used to rank different treatments.

Results

Description of the included studies

The search strategy described in Table 1 and Supplementary Table S1 retrieved 9838 records, including 1121 and 8717 for PDC- and BMSC-related studies, respectively. After removal of duplicates and initial screening based on titles and abstracts, 513 records (78 and 435 for PDC- and BMSC-related studies, respectively) were included for full-text analysis (Fig. 1), of which 101 records (12 and 88 records for PDC- and BMSC-related studies, respectively) were eligible for systematic review. A total of 94 records were retrieved after removing the 6 duplicate references that involved both PDC and BMSC. Among them, 85 studies compared the BMSC-based approach with a bare scaffold; 9 studies compared PDC-based approach with a bare scaffold. Only 3 studies compared all the three approaches (BMSCs, PDCs, and bare scaffold) in one study.

FIG. 1.

Flowchart of the screening process and study selection. The number of studies in each exclusion category were indicated behind each item. WOS, World of Science.

As shown in Tables 2 –4, the characteristics of the included studies varied substantially. Nine different species were used for cell harvest: 26 studies were performed with rats, 21 with rabbits, 15 with humans, 13 with dogs, 7 with goats, 4 with sheep, 4 with mice, 2 with pigs, and 2 with minipigs. The bone marrow aspirates were documented from 5 different tissue origins: 38 studies used femur/tibia, 24 studies used ilium, 2 studies used humerus, 1 study used sternebra, and 23 studies did not specify the origin. In contrast, PDCs were mainly isolated from femur/tibia (8 studies), jaw (3 studies), and calvarium (1 study). Multiple types of cell carriers or biomaterial scaffolds were used, including natural (e.g., collagen and platelet-rich plasma) and synthetic biomaterials. The included synthetic biomaterials were ceramics (e.g., hydroxyapatite, tricalcium phosphate), polymers (e.g., polyglycolic acid), and composites (e.g., apatite-coated silk).

Table 2.

Characteristics of the Included Studies that Compared Bone Marrow Stromal Cells-Based Approach with Bare Scaffold

Reference	Cell source	Tissue origin	Animal model	Implant site	Defect type	Defect size	Scaffold	Cell loading density per scaffold	Scaffold Size	Healing duration	Measurement	Major result
Adamzyk et al⁵⁷	Sheep	Bone marrow aspirate	Sheep	Calvarium	Bicortical	10 mm in diameter	PEKK	1 × 10⁶	10 mm in diameter × 7 mm in height	12 weeks	microCT	BMSC-based<bare scaffold ( NS )
Ai et al⁵⁸	Rabbit	Iliac crest	Rabbit	Tibia	Bicortical	5 mm segmental	Human bone fragment	1 × 10⁶	1 × 1 × 30 mm	4/8 weeks	Histomorphometry	4 weeks: BMSC-based>bare scaffold (NS); 8 w: BMSC-based>bare scaffold^a
Al-Qadhi et al⁵⁹	Rabbit	Bone marrow aspirate	Rabbit	Tibia	Monocortical	6 mm in diameter	Nanobone^®:HA+amorphous silica gel	1 × 10⁶	200 mg granules	2/4/6 weeks	Histomorphometry	2/4 weeks: BMSC-based>bare scaffold (NS); 6 weeks: BMSC-based>bare scaffold^a
Alge et al⁶⁰	Rabbit	Femur	Rabbit	Calvarium	Bicortical	10 mm in diameter	PPF reinforced DCPD	1.5 × 10⁵	9 mm in diameter × 7 mm in height	6 weeks	Histomorphometry	BMSC-based>bare scaffold (NS);
Alhag et al⁶¹	Rat	Femur and tibia	Rat	Calvarium	Bicortical	5 mm in diameter	Collagen–glycos- aminoglycan	6 × 10⁵	16-mm diameter disk (height not mentioned)	7 days	Histomorphometry	BMSC-based>bare scaffold (NS);
Alhag et al⁶²	Rat	Femur and tibia	Rat	Calvarium	Bicortical	5 mm in diameter	Collagen–glycos- aminoglycan	6 × 10⁵	16-mm diameter disk (height not mentioned)	12 weeks	Histomorphometry	BMSC-based<bare scaffold^a
Aloise et al⁶³	Rabbit	Tibia	Rabbit	Calvarium	Bicortical	12 mm in diameter	Bio-Oss	N	N	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Beloti et al⁶⁴	Rat	Femur	Rat	Calvarium	Bicortical	5 mm in diameter	PLGA/cap	2 × 10⁵	5 mm in diameter × 2 mm in height	4 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Ben-David et al⁶⁵	Rat	Femur and tibia	Mouse	Calvarium	Bicortical	5 mm in diameter	Hydrogel	2.5 × 10⁵	5 mm in diameter × 2 mm in height	8 weeks	microCT	BMSC-based>bare scaffold^a
Bölgen et al⁶⁶	Rat	Femur and tibia	Rat	Calvarium	Bicortical	8 mm in diameter	HEMA-Lactate-Dextran cryogel	1 × 10⁶	8 mm-diameter disk (height not mentioned)	30/90/180 days	Histomorphometry	30/90 days: BMSC-based>bare scaffold (NS); 180 days: BMSC-based<bare scaffold (NS);
Bruder et al⁶⁷	Human	Bone marrow aspirate	Rat	Femur	Bicortical	8 mm segmental	HA+beta-TCP	N	4 mm in diameter × 8 mm in height with a 1 mm central canal	4/8/12 weeks	Histomorphometry	4/8 weeks: BMSC-based>bare scaffold (NS); 12 weeks: BMSC-based>bare scaffold^a
Bu et al⁶⁸	Dog	Bone marrow aspirate	Dog	Muscle pocket	N	Ectopic	DBM	N	2.5 × 2 × 1.5 cm	8/12/16 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Burastero et al⁶⁹	Human	Long bone	Rat	Femur	Bicortical	6 mm gap	Natural bone mineral spongiosa granules	0.5, 1, 2, 3 × 10⁶	120 mg granules	2/10/16 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Cao et al⁷⁰	Pig	Iliac Crest	Pig	Calvarium	Monocortical	3 × 1.8 cm oval	HA/TCP	1 × 10⁶	N	6 months	Histomorphometry	BMSC-based>bare scaffold ^*
Chen et al²¹	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9 × 10⁵	6 mm in diameter^*2 mm in height	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Chen et al³³	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9 × 10⁵	6 mm in diameter × 2 mm in height	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Chen et al⁷¹	Rabbit	Femur	Rabbit	Calvarium	Bicortical	15 mm in diameter	Nha; auto-bone	1 × 10⁶	N	3 months	microCT	BMSC-based>bare scaffold (NS)
Chen et al⁷²	Rabbit	Iliac crest	Rabbit	Calvarium	Bicortical	15 mm in diameter	GGT; GGT-GSB	1 × 10⁶	15 mm in diameter × 2 mm in height	1/2/4 weeks	Histomorphometry	GGT 1 week: BMSC-based>bare scaffold (NS); GGT 2/4 weeks, GGT-GSB 1/2/4 weeks: BMSC-based>bare scaffold^a
Chen et al⁷³	Rat	Femur and tibia	Rat	Femural head	Monocortical	4 mm in diameter × 5 mm in depth	N-HA/PU40	2 × 10⁵	10 mm in diameter × 2 mm in height	4 weeks	microCT	BMSC-based>bare scaffold^a
Chen et al⁷⁴	Human	N	Rat	Calvarium	Bicortical	8 mm in diameter	CPC	3 × 10⁵	8 mm in diameter × 1 mm in height	4/12/24 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Chu et al⁷⁵	Goat	N	Goat	Tibia	Bicortical	3 cm segmental	Beta-TCP	N	N	6 months	Histomorphometry	BMSC-based>bare scaffold^a
Coelho de Faria et al⁷⁶	Rabbit	Tibia	Rabbit	Calvarium	Bone augmentation	6 mm in diameter × 5 mm in height	Bio-Oss	N	N	56 days	Histomorphometry	BMSC-based>bare scaffold (NS)
Darnell et al³²	Human	Product	Rat	Calvarium	Bicortical	8 mm in diameter	Alginate Hydrogel	1 × 10⁷/ml hydrogel	N	3 months	microCT	BMSC-based>bare scaffold (NS)
De Kok et al⁷⁷	Rat	Femur	Rat	Calvarium	Bicortical	8.9 mm in diameter	Collagen	N	N	28 days	Histomorphometry	BMSC-based>bare scaffold^a
De Santana Santos et al⁷⁸	Rat	Femur	Rat	Calvarium	Monocortical	5 mm in diameter	Geletin sponge	5 × 10⁵/mg	N		microCT	BMSC-based>bare scaffold (NS)
Dumas et al⁷⁹	Mouse	Medullary cavity	Mouse	Calvarium	Bicortical	4 mm in diameter	Xenograft	3 × 10⁵	4 mm in diameter × 0.3 mm in height	2/8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Elkhateb et al⁸⁰	Rat	Femur and tibia	Rat	Calvarium	Bicortical	5 mm in diameter	Propylene mesh; acellular dermal graft	1 × 10⁵	5 mm-diameter piece (height not mentioned)	1 month	Histomorphometry	BMSC-based>bare scaffold^a
Eniwumide et al³¹	Goat	Iliac crest	Goat	Musclle pocket	N	Ectopic	Allograft	1 × 10⁷/mL	N	12 weeks	Histomorphometry	BMSC-based>bare scaffold (NS)
Frasca et al⁸¹	Rat	Femur	Rat	Femur	Monocortical	3 mm in diameter	BCP ceramic granules; hydrogel	5 × 10⁵	6 mm in diameter × 1 mm in height	7/30/90 days	microCT	BMSC-based>bare scaffold (NS)
Gallego et al⁸²	Sheep	Iliac crest	Sheep	Mandible	Bicortical	3 cm segmental	Fibrin clot	2–3 × 10⁷	30 × 15 × 10 mm	12/32 weeks	microCT	BMSC-based>bare scaffold^a
Gao et al⁸³	Rat	Femur and tibia	Rat	Calvarium	N	8 mm in diameter	N-HA/PA6	N	8 mm-diameter piece (height not mentioned)	4/8/16 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Ghaffar et al⁸⁴	Dog	Femur	Dog	Jaw	Monocortical	N	Polymer	4.7 × 10⁶	N	1.5/3 months	Histomorphometry	BMSC-based>bare scaffold^a
He et al⁸⁵	Rat	Femur and tibia	Rat	Calvarium	Bicortical	8 mm in diameter	CAH	1 × 10⁶	N	12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
He et al⁸⁶	Rat	Femur	Rat	Muscle pocket	N	Ectopic	Chitosan hydrogel	1 × 10⁵	N	2/4/6/8/12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
He et al⁸⁷	Rat	Femur	Rat	Muscle pocket	N	Ectopic	Nha-CS	1 × 10⁵	N	2/4/6/8/12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Hu et al⁸⁸	Rabbit	N	Rabbit	Radius	Monocortical	1.5 cm segmental	BCP; nHA-BCP	428 ± 46; 562 ± 37/mm²	N	12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Hu et al⁸⁹	Dog	N	Dog	Jaw	Monocortical	5 × 5 × 6 mm	CPC	5 × 10⁴	5 × 5 × 6 mm	4/8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Huang et al⁹⁰	Rat	Tibia and femur	Rat	Femur	Monocortical	5 mm	PLGA-GO	N	N	2/4/8 weeks	microCT t; histomorphometry	4 weeks: BMSC-based>bare scaffold (NS);8 weeks: BMSC-based>bare scaffold^a
Ingavle et al²⁸	Sheep	Sternebra	Sheep	Iliac crest	Bicortical	15 mm in diameter × 5 mm in depth	Alginate and hyaluronate-based polymers	5 × 10⁵	8 mm in diameter (height not mentioned)	12 weeks	microCT	BMSC-based>bare scaffold^a
Jo et al⁹¹	Human	N	Nude mouse	Radius	Bicortical	6 mm segmental	HA; TCP	7.5 × 10⁶	6 mm long, 4 mm in external diameter, 1 mm in internal diameter	12 weeks	microCT	Osteogenic medium pretreatment: BMSC-based>bare scaffold^a;Non-pretreatment: BMSC-based>bare scaffold (NS)
Kanczler et al⁹²	Human	N	MF-1 nu/nu mouse	Radius	Bicortical	5 mm segmental	PLA/VEGF	2 × 10⁵	N	4 weeks	microCT	BMSC-based<bare scaffold (NS)
Kargozar et al⁹³	Human	Iliac crest	Rat	Calvarium	N	7 mm in diameter	Bioactive glass/gelatin	N	N	4/12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Kasten et al⁹⁴	Rabbit	N	Rabbit	Radial diaphysis	Monocortical	15 mm segmental	CDHA; CDHA+PRP	5 × 10⁶	4 mm in diameter × 15 mm in height	16 weeks	microCT	CDHA: BMSC-based>bare scaffold^a;CDHA+PRP: BMSC-based>bare scaffold (NS)
Kempen et al⁹⁵	Goat	Iliac crest	Goat	Subcutaneous Pocket	N	Ectopic	Poly(propylene fumarate)	5 × 10⁵	6 mm in diameter × 3 mm in height	9 weeks	Histomorphometry	BMSC-based<bare scaffold ( NS )
Khojasteh et al⁹⁶	Dog	Humerus	Dog	Mandible	Monocortical	20 × 10 × 10mm	PCL-TCP	5 × 10⁵	20 × 10 × 10 mm	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Khojasteh et al⁹⁷	Dog	Humerus	Dog	Jaw	Bicortical	10 mm in diameter	Beta-TCP-PLGA	5 × 10⁵	12 × 6 × 8 mm	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Kim et al⁹⁸	Human	N	Rat	Calvarium	Bicortical	8 mm in diameter	HA-based hydrogel	8 × 10⁵	50 μL	4 weeks	microCT	+BMP2: BMSC-based>bare scaffold^a;-BMP2: BMSC-based<bare scaffold (NS)
Kim et al⁹⁹	Rat	Femur and tibia	Rat	Calvarium	Bicortical	5 × 5 mm	Small intestine submucosa sponge	5 × 10⁵	5 × 5 mm	2/4 weeks	Histomorphometry	BMSC-based<bare scaffold (NS)
Kong et al¹⁰⁰	Rat	Femur and tibia	Rat	Calvarium	Bicortical	5 mm in diameter	Sodium alginate+PLLA	5 × 10⁵/2 mL	N	4/8 weeks	microCT	BMSC-based>bare scaffold^a
Kruyt et al¹⁰¹	Goat	Iliac wing	Goat	Intramuscular and iliac wing	Monocortical	17 mm in diameter	BCP	2.98 × 10⁶	7 × 7 × 7 mm	9/12 weeks	Histomorphometry	Orthotopic: BMSC-based>bare scaffold (NS);Ectopic: BMSC-based>bare scaffold^a
Lee et al¹⁰²	Rat	Femur	Rat	Calvarium	Bicortical	8 mm in diameter	DBM	N	N	12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Lee et al¹⁰³	Rat	Femur and mandible	Rat	Calvarium	Bicortical	8 mm in diameter	Collagen-based sponge	5 × 10⁶	10 mm in diameter × 4 mm in thickness	8 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Lee et al²⁹	Rabbit	Femur and tibia	Rabbit	Calvarium	Bicortical	6 mm in diameter	Bio-Oss	10⁶	N	3/6 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Leotot et al¹⁰⁴	Human	Iliac crest	Mouse	N	Bone augmentation	N	Beta-TCP/HA	10⁶	N	7 weeks	microCT; histomorphometry	BMSC-based>bare scaffold ^*
Lin et al¹⁰⁵	Rabbit	Ilium	Rabbit	Femur	Bicortical	5 mm in diameter × 10mm in height	NanoHA-type I collagen bead	5 × 10⁵	6 mm in diameter × 10 in height	4/8 weeks	microCT	BMSC-based>bare scaffold^a
Lisignoli et al¹⁰⁶	Rat	Femur	Rat	Radius	Bicortical	5 mm segmental	Hyaluronic acid-based polymer	2 × 10⁶/cm²	5 × 7 × 2 mm	40/80/160/200 days	Histomorphometry	BMSC-based>bare scaffold^a
Liu et al¹⁰⁷	Goat	Ilium	Goat	Tibia	Bicortical	25 mm segmental	nHA/collagen/PLLA/chitin	2 × 10⁶	8.5 mm in diameter × 5 mm in height	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Long et al¹⁰⁸	Mouse	Femur and tibia	Mouse	Femur	Bicortical	4mm segmental	Devitalized allograft	10⁶	4 mm in height (diameter not mentioned)	2/4/6 weeks	microCT	BMSC-based>bare scaffold^a
Lovati et al¹⁰⁹	Sheep	Iliac crest	Sheep	Femur and tibia	Monocortical	8 mm in diameter × 4 mm in depth	HA-based scaffolds	2 × 10⁶	8 mm in diameter × 4 mm in height	2/4 months	Histomorphometry	BMSC-based<bare scaffold (NS)
Ma et al¹¹⁰	Rabbit	Ilium	Rabbit	Calvarium	Bicortical	15 mm in diameter	DBM	2.2 × 10⁷	15 mm in diameter × 2 mm in height	6/12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Mylonas et al¹¹¹	Dog	N	Dog	Mandible	Monocortical	5 mm in diameter × 8 mm in depth	TCP/Bioglass	5 × 10⁶/ml	N	4/7 weeks	Histomorphometry	BMSC-based>bare scaffold (NS)
Nair et al¹¹²	Goat	N	Goat	Femur	Bicortical	20 mm segmental	HA-Si	1 × 10⁵/cm²	2-cm outer diameter and 7-mm inner diameter × 2 cm height	2 months	Histomorphometry	BMSC-based>bare scaffold^a
Naudot et al¹²	Rat	Femur	Rat	Calvarium	Bicortical	5mm in diameter	PCL-nHA	1 × 10⁶/cm²	N	8 weeks	microCT	2/4/6 weeks: BMSC-based>bare scaffold (NS);8 weeks: BMSC-based>bare scaffold^a
Osugi et al¹¹³	Human	N	Rat	Calvarium	Bicortical	5 mm in diameter	Agarose gel	1 × 10⁵	N	8 weeks	microCT	BMSC-based>bare scaffold^a
Pieri et al¹¹⁴	Minipig	Iliac crest	Minipig	Mandible	Monocortical	3.5 mm diameter × 8 mm depth	Fluorohydroxyapatite/platelet gel/collagen	4 × 10⁷/5 ml	N	3 months	Histomorphometry	BMSC-based>bare scaffold^a
Qiu et al¹¹⁵	Minipig	Iliac crest	Minipig	Femur	Monocortical	8 mm in diameter × 10 mm in height	CPC	1 × 10⁶/	8 mm in diameter × 10 mm in height	6/12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Saad et al¹¹⁶	Rabbit	Femur	Rabbit	Mandible	Bicortical	10 × 15 mm	Beta-TCP	1–1.46 × 10⁷	N	2/4/12/24 weeks	Histomorphometry	2 weeks: BMSC-based<bare scaffold^a;4/12/24 weeks: BMSC-based>bare scaffold (NS)
Stockmann et al³⁷	Pig	Tibia	Pig	Calvarium	Monocortical	10 mm in diameter × 10mm in depth	Collagen	2 × 10⁶	N	7/14/30/60/90 days	Histomorphometry	7 days: BMSC-based<bare scaffold (NS);14/30 days: BMSC-based>bare scaffold (NS);60/90 days: BMSC-based>bare scaffold^a
Sun et al¹¹⁷	Rabbit	Fibula	Rabbit	Calvarium	Monocortical	8 × 4 mm	Osteobone	2 × 10⁷/ml	N	2/4/8 weeks	Histomorphometry	BMSC-based>bare scaffold (NS)
Tao et al¹¹⁸	Rabbit	Iliac crest	Rabbit	Subcutaneou	N	Ectopic	Artificial bone	N	N	4/8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Thorpe et al¹¹⁹	Rat	Femur and tibia	Rat	Femur	Monocortical	1 mm in diameter	HA nanoparticles + hydrogel	2 × 10⁶/ml	N	4 weeks	microCT; histomorphometry	BMSC-based<bare scaffold (NS)
Timashev et al¹²⁰	Mouse	N	Mouse	Calvarium	N	4 mm in diameter	Star-shaped PLA	2 × 10⁵	N	5/10 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Tu et al¹²¹	Rat	Femur and tibia	Rat	Calvarium	Bicortical	6 mm in diameter	PLA/HA	N	6 mm in diameter × 0.6 mm in height	12 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Wang et al²⁷	Dog	Iliac crest	Dog	Mandible	Monocortical	5 × 5 × 6 mm	Beta-TCP	N	N	12 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Wang et al¹²²	Human	N	Rabbit	Mandible	Monocortical	10 × 5 × 3 mm	Porous Nano-HA/Collagen/PLA Scaffold	5 × 10⁶	10 × 5 × 3 mm	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Xia et al¹²³	Rabbit	N	Rabbit	Maxilla	Monocortical	8 × 4 mm	CPC	N	N	2/4/8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Xu et al¹²⁴	Rat	Femur	Rat	Calvarium	Bicortical	5 mm in diameter	Beta-TCP	4 × 10⁶	5 mm in diameter × 2 mm in height	8 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Xu et al¹²⁵	Dog	Femur	Dog	Mandible	Monocortical	6 × 4 × 5 mm	Beta-TCP	6 × 10⁶	0.3mL particles	12 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Xuan et al¹²⁶	Dog	Iliac crest	Dog	Sternal	Bicortical	3 × 2.5 cm segmental	PCL/HA	N	9 × 7 × 5 mm	3/6/9 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Yang et al³⁰	Rat	N	Rat	Mandible	Monocortical	1 × 3 mm	Microcarrier gelatin beads	N	N	3 weeks	Histomorphometry	BMSC-based<bare scaffold^a
Yu et al¹²⁷	Dog	Iliac crest	Immunodeficient rat	Subcutaneous pocket and calvarium	Bicortical	4 mm in width	Bio-Oss	N	N	8 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Zhang et al¹²⁸	Dog	Iliac crest	Dog	Mandible	Bicortical	10 × 5 × 15mm	Beta-TCP	N	N	4/12/20 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Zhang et al¹²⁹	Rabbit	N	Rabbit	Ulna	Bicortical	15 mm segmental	PLGA	1.9 × 10⁶	4mm in diameter × 15mm in height	6/12 weeks	microCT; histomorphometry	BMSC-based>bare scaffold^a
Zong et al¹³⁰	Human	N	Rat	Calvarium	Bicortical	5 mm in diameter	PLGA	5 × 10⁵	10mm in diameter × 3mm in height	10/20 weeks	Histomorphometry	BMSC-based>bare scaffold^a
Zou et al¹³¹	Goat	Iliac crest	Goat	Jaw	Monocortical	3 mm diameter × 3 mm in depth	CPC	N	3 mm in diameter × 3mm in height	3 months	microCT	BMSC-based>bare scaffold (NS)

p < 0.05.

BCP, biphasic calcium phosphate; CAH, chitosan/alginate/HA complex scaffold; CDHA, calcium-deficient hydroxyapatite; CPC, calcium phosphate cement; DBM, demineralized bone matrix; DCPD, dicalcium phosphate dehydrate; GO, graphene oxide; GSB, Gu-Sui-Bu; HA, hydroxyapatite; MMP, matrix metalloproteinase; N, not mentioned; NS, no significant difference; PA6, polyamide 6; PCL, polycaprolactone; PEKK, polyetherketoneketone; PLGA, poly(lactide-co-glycolide); PPF, poly(propylene fumarate); TCP. tricalcium phosphate; “>” or “<” represents that the bone-forming efficacy of BMSC-based approach is higher or lower than bare scaffold.

Table 3.

Characteristics of the Included Studies that Compared Periosteum-Derived Cells-Based Approach with Bare Scaffold

Reference	Cell source	Tissue origin	Animal model species	Implant site	Defect type	Defect size	Scaffold	Cell loading density per scaffold	Scaffold Size	Healing duration	Measurement	Major result
Bakker et al¹³²	Rabbit	Tibia	Rabbit	Tibia	Monocortical	2 cm	HA; Ti; PH70αTCP	2 × 10⁷	6 mm in diameter × 20 mm in height	10 weeks	Histomorphometry	PDC-based>bare scaffold (NS)
Chen et al²¹	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9 × 10⁵	6 mm in diameter × 2mm in height	8 weeks	Histomorphometry	PDC-based>bare scaffold^a
Chen et al³³	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9^*10⁵	6 mm in diameter × 2mm in height	8 weeks	Histomorphometry	PDC-based>bare scaffold^a
Katagiri et al²⁰	Mouse	Femur and tibia	Mouse	Subcutaneous pocket	Ectopic	N	BioOss; Copios	1.7 × 10⁶	3 mm in diameter × 3–5mm height	6 weeks	microCT	PDC-based>bare scaffold (NS)
Maréchal et al¹³³	Rabbit	Tibia	Rabbit	Skull	Bone augmentation	N	TCP; HA/TCP	N	TCP: 10 × 8 × 6 mmHA/TCP: 5 mm^5 mm^5 mm	12 weeks	Histomorphometry	PDC-based<bare scaffold (NS)
Miyamoto et al³⁴	Rabbit	Tibia	Rabbit	Calvarium	Monocortical	6 mm in diameter	PLLA + collagen	1 × 10⁶	4.5 mm- diameter and 3.5 mm- height dome	12 weeks	Histomorphometry	PDC-based>bare scaffold^a
Moreira-Gonzalez et al¹³⁴	Rabbit	Tibia	Rabbit	Calvarium	Bicortical	15 mm in diameter	Bioglass	N	N	12 weeks	Histomorphometry	PDC-based>bare scaffold^a
Paulo et al²²	Rat	Calvarium	Rat	Cavalrium	Bicortical	8 mm in diameter	HA-collagen xenograft	1 × 10⁵	8 in diameter × 1.5 mm in height	30/90 days	Histomorphometry	30 days: PDC-based<bare scaffold (NS);90d: PDC-based<bare scaffold^a
Stockmann et al³⁷	Pig	Tibia	Pig	Calvarium	Monocortical	10 mm in diameter × 10mm in depth	Collagen	2 × 10⁶	N	7/14/30/60/90 days	Histomorphometry	7/14 days: PDC-based<bare scaffold (NS);30/60 days: PDC-based>bare scaffold (NS);90 days: PDC-based>bare scaffold^a

p < 0.05.

Table 4.

Characteristics of the Included Studies that Directly Compared Bone Marrow Stromal Cells-Based with Periosteum-Derived Cells-Based Approach

Reference	Cell source	Tissue origin	Animal model	Implant site	Defect type	Defect size	Scaffold	Cell loading density per scaffold	Scaffold Size	Healing duration	Measurement	Major result
Chen et al²¹	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9 × 10⁵	6 mm in diameter × 2mm in height	8 weeks	Histomorphometry	PDC-based>BMSC-based (NS)
Chen et al³³	Human	Limb	Mouse	Subcutaneous pocket	Ectopic	N	Beta-TCP	9 × 10⁵	6 mm in diameter × 2mm in height	8 weeks	Histomorphometry	PDC-based>BMSC-based(NS)
Jaquiéry et al³⁶	Human	PDC: jaw; BMSC: iliac crest	Mouse	Subcutaneous pocket	Ectopic	N	Bio-Oss; Vitoss	N	Vitoss: 6 × 6 × 6 mm	8 weeks	Histomorphometry	PDC-based<BMSC-based^a
Ribeiro et al³⁸	Dog	PDC: mandible; BMSC: iliac crest	Dog	Alveolar bone	Monocortical	4 × 5 mm	Collagen sponge	2 × 10⁷	N	3 months	Histomorphometry	PDC-based>BMSC-based (NS)
Stockmann et al³⁷	Pig	Tibia	Pig	Calvarium	Monocortical	10 × 10 mm	Collagen	2 × 10⁶	N	7/14/30/60/90 days	Histomorphometry	PDC-based<BMSC-based (NS)
Zhu et al³⁵	Dog	PDC: mandible; BMSC: iliac	Dog	Subcutaneous pocket	Ectopic	N	Fibrin glue	1 × 10⁶	400 μL glue	12 weeks	Histomorphometry	PDC-based>BMSC-based^a

p < 0.05.

The total cell-loading number ranged from 10⁵ cells to 4 × 10⁷ cells per scaffold, and the cell-loading density ranged from 0.13 × 10⁴ to 10 × 10⁴ cells/mm³ (if the volume of the scaffold was mentioned). The obtained cell-laden constructs were evaluated for bone formation via either ectopic or orthotopic implantation. The ectopic evaluations were mainly performed in subcutaneous pockets (8 studies) or muscles (1 study). The orthotopic evaluations were performed on a variety of defects, including the calvaria defect (40 studies), jaw defect (16 studies), femoral/tibial defect (16 studies), radial/ulnar defect (6 studies), iliac defect (2 studies), and sternal defect (1 study). The orthotopic defects were categorized as bicortical (49 studies) if they penetrate both layers of cortical bone. Otherwise they were categorized as monocortical (28 studies).

The animal models used for evaluation included rat (31 studies), rabbit (22 studies), dog (13 studies), mouse (11 studies), goat (7 studies), pig (4 studies), and sheep (4 studies).

Regarding the outcome measures, both histomorphometry and microCT were used to quantify the bone-forming efficacy, and the results of individual documentation varied (Table 2–4). Notably, 29 out of 85 studies compared BMSC-based approach with the bare scaffold based on microCT analysis, and 21 of them showed a significant superiority of BMSC-based approach over the bare scaffold (Table 2). In comparison, only one study compared PDC-based approach with the bare scaffold using microCT, and no significant difference was observed (Table 3). Furthermore, no studies used microCT as outcome measurements to directly compare BMSC with PDC-based approach.

Hence, only histomorphometric new bone formation outcome measurement was chosen for further meta-analysis. Based on histomorphometry, 52 out of 66 studies demonstrated a significant superiority of the BMSC-based approach over the bare scaffold group (Table 2). Five out of eight studies showed a higher bone-forming efficacy of the PDC-based approach compared with the bare scaffold group (Table 3). Notably, among the six studies that directly compared the PDC-based approach with the BMSC-based approach, four studies showed no significant difference between these two types of cells, whereas the other two studies revealed the superiority of either BMSCs or PDCs over their counterpart (Table 4).

Risk of bias and quality of reporting

Figure 2 shows the overall results of the risk of bias assessment of the 94 studies included in this systematic review. Regarding the selection bias, 59.57% of the included studies reported randomization across the experiment. However, only 18.09% stated the application of allocation sequence, and only 8.51% mentioned allocation concealment. Of the included studies, 72.34% stated similar baseline characteristics between experimental and control groups. In addition, 68.08% and 89.36% of the included studies were scored as under unclear risk of bias concerning the performance bias items “random housing” and “blinding,” respectively.

FIG. 2.

Risk of bias evaluated using an adapted version of SYRCLE's risk of bias tool. The risk of bias of each study was rated as “high,” “low,” or “unclear.” Color images are available online.

For the detection bias item “random outcome assessment,” 100% of the included studies were scored as low risk of bias, because the outcome of invention and control groups were assessed at the same time points. Only 21.28% of the studies reported that outcome assessment was blinded based on item 7. For attrition bias, 95.74% of the studies were scored as low risks of bias, as they adequately addressed incomplete outcome data.

Pairwise meta-analysis of outcome measures

A total of 66 studies were included for a pairwise meta-analysis of the BMSC-based approach vs the bare scaffold group. BMSC-based approach showed significant higher efficacy (SMD and 95% CI: 2.59 [2.11–3.07]; I² = 87.26%, p < 0.001) than the bare scaffold group (Fig. 3). Subgroups analysis showed a beneficial effect of BMSCs in all animal species model except sheep. Interestingly, the SMDs in dog and mouse subgroups were higher than in other groups. Regarding PDCs, the pairwise meta-analysis revealed no statistically significant difference between PDCs and a bare scaffold on the basis of a total of 8 studies (SMD and 95% CI: 0.48[−0.16 to 1.13]; I² = 75.42%, p < 0.001) (Fig. 4).

FIG. 3.

Forest plot of the studies that compared BMSC-based approach with bare scaffold group in the pairwise meta-analysis. The forest plot displays relative weight of individual experiments, the SMD, and 95% CI. The diamond indicates the global estimate and its 95% CI. BMSC, bone marrow stromal cells; CI, confidence interval; SMD, standardized mean difference. Color images are available online.

FIG. 4.

Forest plot of the studies that compared the PDC-based approach with the bare scaffold group in the pairwise meta-analysis. The forest plot displays relative weight of individual experiments, the SMD, and 95% CI. The diamond indicates the global estimate and its 95% CI. Color images are available online.

It is noted that substantial variation of the results existed in different animal models. Favorable effects of PDCs were only observed in mouse and pig models. We further performed a pairwise meta-analysis of the six studies involving a direct comparison between the PDC-based approach and the BMSC-based approach. Given the small number of studies, there was no significant difference between these two groups (SMD and 95% CI: −0.79[−2.76 to 1.18]; I² = 95.57%, p < 0.001; Fig. 5). Furthermore, slight favorable effect of the PDC-based approach showed over MSC-based approach was observed in dog models.

FIG. 5.

Forest plot of the studies that directly compared the PDC-based approach with the BMSC-based approach in the pairwise meta-analysis. The forest plot displays the relative weight of individual experiments, the SMD, and 95% CI. The diamond indicates the global estimate and its 95% CI. Color images are available online.

Among the funnel plots of the three pairwise meta-analyses, the studies that compared the BMSC-based approach with bare scaffold showed obvious underrepresentation of the small, negative studies (Supplementary Fig. S1a). The funnel plots of the other two pairwise meta-analyses appeared to lack studies of large sample size (Supplementary Fig. S1b, c).

Network meta-analysis

To address the issue of a lack of sufficient studies with direct evidence, the network analysis was further performed to compare the BMSC-based approach to the PDC-based approach, which consisted of three arms, namely: the BMSC-based approach, the PDC-based approach, and the bare scaffold group (Fig. 6a). By doing this, an indirect comparison between BMSC-based and PDC-based approaches can be achieved through the common comparator “the bare scaffold group.”^45,46 Furthermore, network meta-analysis can combine direct evidence (studies that directly compared BMSC-based and PDC-based approaches) with indirect evidence (studies that compared them via “the bare scaffold group”) to calculate a mixed effect size, hence facilitating an increased amount of evidence.⁴⁶

FIG. 6.

Results of network meta-analysis. (a) Network geometry of the network meta-analysis of the comparisons of histomorphometric new bone formation. The number on the spheres represented the number of the studies that included the group as an intervention. The number on the line that connected the spheres represented the number of comparisons between these two groups. (b) Interval plot of random-effect network meta-analysis evaluating the difference in histomorphometric new bone formation. A mean difference with a 95% CI was shown. (c) SURCA ranking result of network meta-analysis. (d–f) Cumulative probability of ranking evaluating the histomorphometric new bone formation of the BMSCs (d), PDCs (e), and bare scaffold (f), respectively. PDC, periosteum-derived cells. Color images are available online.

Our results suggested that no obvious heterogeneity (I² = 0.4%) and no obvious publication bias (Supplementary Fig. S2) were detected between comparisons. Furthermore, no significant inconsistency in our analyses was detected based on the results of consistency test using global inconsistency and loop inconsistency model (Supplementary Tables S2, S3).

Our data revealed that compared with the bare scaffold group, both the BMSC-based approach (OR = 13.32, 95% CI: 10.40–16.25) and the PDC-based approach (OR = 10.88, 95% CI: 3.65–18.11) significantly enhanced new bone formation (Fig. 6b). However, no significant difference was found between these two types of cell-based approaches (OR = −2.452, 95% CI: −9.84 to −4.85), which was consistent with the aforementioned pairwise meta-analysis in Result 3. Furthermore, SUCRA values showed that the BMSC-based approach was most likely (74.2%) to be ranked the best among all the three interventions, while the probability to be ranked best of PDC-based approach and bare scaffold were 25.8% and 0.0%, respectively (Fig. 6c–f).

Discussion

This review aimed to systematically evaluate the bone-forming efficacy of the BMSCs- and PDCs-based approaches. The result of this systematic review and pairwise meta-analysis revealed that the BMSCs-based approach had a favorable effect on new bone formation, as displayed by the positive effect on histomorphometric analysis. However, BMSCs-based and PDC-based approaches had no significant difference. Network meta-analysis was further conducted to increase the amount of evidence to compare these two cell-based approaches. The results suggest that both BMSC and PDC can improve new bone formation compared with the bare scaffold. Although OR and 95% CI showed no significant difference between BMSC and PDC, SURCA ranking suggested that BMSC had a higher probability to be ranked better than PDC.

The currently used approach of systematic review allowed the inclusion of a sufficient number of studies, which involved BMSCs- or PDCs-based approaches for bone regeneration. By doing this, we could perform the meta-analysis and explore the effect in different subgroups. However, there are some potential limitations related to this approach. First, due to the nature of animal studies, experimental variabilities existed regarding the animal species, tissue origin of seed cells, animal model species, implant site, defect type and size, and so on, which, not surprisingly, leads to substantial statistical heterogeneity.^47,48 To account for the expected heterogeneity, a random effect model was used in the pairwise meta-analysis. Also, subgroups analyses regarding animal model species were performed to explain the heterogeneity.

However, the subgroup analyses did not notably reduce the heterogeneity except in the PDC-based approach vs bare scaffold. Notably, as shown in funnel plots, pairwise meta-analyses were subject to publication bias, which can be caused by either nonpublication of negative results, small sample size, or other factors, including true study heterogeneity or differences in study quality.⁴⁹ Finally, the study quality assessment revealed poor reporting quality of animal studies existed in the evaluation of cell-based approaches. Most items in the assessment were rated as unclear because they were not mentioned in the articles.⁴¹ To avoid bias, randomization and blindness are strongly suggested for preclinical and clinical studies. However, only 18.08% of studies reported the generation of a random allocation sequence. Also, only 21.2% of the studies reported blindness across the experiment. Therefore, the results of our systematic review and meta-analysis might cause overestimates of certain interventions.

Despite these limitations, the combined analysis of the included studies still generated extra and valuable information that could not be derived from individual studies.

One of the most important issues for the clinical application of cell-based approaches is the type of cell used. As shown in this systematic review, BMSCs have been explored extensively in both preclinical evaluations for bone regeneration, while the studies that explored PDCs-based approaches are rather limited. Currently, many systematic reviews were conducted focusing on the bone regeneration capacity of BMSCs or bone marrow aspirates,^50–54 most of which showed promising results of BMSC-based cell therapy in treating long bone reunions,⁵⁰ osteoporosis,⁵³ and osteoarthritis.⁵⁴ However, these studies either included a variety of bone marrow-derived products^50,51 or only evaluate in a certain disease model.^53,54 No systematic review and meta-analysis of the bone regeneration capacity of BMSC-based approach in bone defects was conducted.

Meanwhile, PDC was an emerging source for bone tissue engineering, and there is currently no systematic review regarding its bone-forming efficacy, let alone the comparison between BMSCs and PDCs. In our systematic review, 64 out of 85 studies reported a positive effect of BMSCs on bone formation. For PDCs, five out of nine demonstrated a statistically significant improvement in new bone formation. The result of the meta-analysis revealed that BMSC-based approach had significantly higher bone-forming efficacy than the bare scaffold. However, no significant difference was found between the PDC-based approach and bare scaffold and the PDC-based approach and bare scaffold. This result is probably due to the limited study number and the obvious heterogeneity (I² = 75.42%, p < 0.001; I² = 95.57%, p < 0.001) within the included studies.

To assess the possible influence of variables on cell-based approach, subgroup analysis regarding animal model species was performed. For BMSC-based approach, rabbit and rat were the most frequently used models, which both showed similar results with the overall effect. Interestingly, studies using mouse as animal model showed higher SMD than other species, which suggested that caution must be taken before scaling preclinical studies up to large animal models because mouse model might overestimate the bone-forming efficacy of cell-based approach. Similar implication can also be obtained from the subgroup analysis of PDC-based approach versus bare scaffold.

To compare the bone-forming efficacy between BMSCs and PDCs, we performed a network analysis to minimize the bias that caused by the limited number of PDCs-related studies.^45,46 Network meta-analysis can integrate the effect size of both direct and indirect evidences, providing more comparisons between interventions.⁴⁵ Currently, this powerful tool has been used to compare of tissue regeneration capacity between different stem cells.⁵⁵ The result of heterogeneity assessment and inconsistency evaluation suggested the successful establishment of the network meta-analysis model. Our network meta-analysis showed that both BMSCs and PDCs showed a comparable positive effect on new bone formation, although BMSCs had a higher ranking probability than PDCs. Therefore, our results indicated that both BMSCs- and PDCs-based approaches can be applied for bone regeneration, and their actual implementation in clinical practice could be based on the specific condition of each individual patient (e.g., accessibility of cells and health condition of donor tissues).⁵⁶

Conclusion

The current systematic review and meta-analysis indicate that both BMSCs- and PDCs-based therapies have a positive effect on in vivo bone formation compared to scaffold-only therapies. Moreover, there is no statistically significant difference between BMSCs and PDCs regarding in vivo bone-forming efficacy, although BMSCs have a higher probability to be ranked better than PDCs. These results provide important information for the implementation of cell-based approaches in clinical practice as a routine treatment in the future.

Footnotes

Acknowledgments

The authors thank Professor Yukang Tu from National Taiwan University and Professor Fang Hua from Wuhan University for their kind help in the methodology of this research.

Authors' Contributions

J.Z.: Methodology, Formal analysis (equal), Investigation (equal), Data curation (equal), Writing—original draft, and Visualization (equal). J.X.: Formal analysis (equal), Investigation (equal), Data curation (equal), Draft editing, and Visualization (equal). W.J.: Conceptualization, Methodology, Writing—Review and Editing, Supervision, Project administration, and Funding acquisition.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study is funded by the National Natural Science Foundation of China (No. 82170931), the Natural Science Foundation of Hubei Province (No. 2020CFB618), and the Fundamental Research Funds for the Central Universities (No. 2042022kf1172).

Supplementary Material

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