GREM1 acts in leptin receptor-expressing skeletal cells to mediate peri-implant fibrosis

Domain: biology
Published: 2026-03-23
Editorial outcome: major_revision

Abstract
Globally, approximately 1.3 million total joint implants are placed annually, and around 80,000 patients develop aseptic loosening, a leading cause of implant failure driven by peri-implant fibrosis. Here, we show that the BMP-antagonist Gremlin-1 (GREM1), expressed by leptin receptor–expressing skeletal (LEPR⁺) cells, is a key regulator of this process. GREM1 is highly expressed by LEPR⁺ cells in peri-implant fibrotic tissue in mice and humans. Conditional deletion of Grem1 in LEPR⁺ cells attenuate peri-implant fibrosis and enhances peri-implant osteogenesis. Transcriptomic and functional analyses show that loss of Grem1 in LEPR⁺ cells upregulate the bone morphogenetic protein (BMP) and WNT pathways, increasing in vivo osteogenesis and reducing fibrous tissue formation. As proof-of-concept, intra-articular administration of a neutralizing antibody against GREM1 (anti-GREM1) in mice prevents and reverses peri-implant fibrous tissue while promoting peri-implant bone formation. Inhibition of GREM1 in LEPR⁺ cells therefore represent a promising strategy to prevent and treat aseptic loosening.

Introduction
Around 1.3 million total joint implants1,2 are implanted worldwide annually for the treatment of osteoarthritis, inflammatory arthritis, post-traumatic arthritis, and avascular necrosis. Peri-implant fibrosis is one of the most common causes of failure in prosthetic joint implants3,4, affecting approximately 80,000 people in the United States with joint prostheses4,5. In peri-implant fibrosis, fibrous tissue forms around the implant instead of bone, preventing proper stabilization of the implant, leading to instability and pain6-8. Addressing this issue typically requires surgical revision, which involves removing the existing implant and surrounding fibrotic tissue, followed by the placement of a new implant. Unfortunately, revision surgeries are associated with significant morbidity, mortality, decreased patient-reported outcomes scores, and increased re-revision rates9-12. Therefore, identifying biological therapeutic targets that can prevent or reverse peri-implant fibrosis is a critical priority to minimize the need for revision surgeries.

We have recently demonstrated that skeletal cells expressing the leptin receptor (LEPR+) are both necessary and sufficient to induce peri-implant fibrosis13. These LEPR+ cells highly overlap with CXCL12+ cells, which are commonly referred to as CXCL12-abundant reticular (CAR) cells14. In addition to CXCL12, LEPR+ cells also express high levels of the transcription factors EBF315 and FOXC116, as well as previously reported markers of skeletal progenitors, including CD20017, PDGFRα18, and alpha-smooth muscle actin (αSMA)19. LEPR+ cells consist of multiple subtypes, including an osteogenic subtype, sometimes referred to as osteoCAR cells, characterized by the expression of alkaline phosphatase (ALPL)20 and an adipogenic subtype, sometimes referred to as adipoCAR cells or marrow adipogenic lineage precursors (MALPs), marked by the expression of adiponectin (Adipoq)20-22. Under surgical stress, CAR cells can become pro-fibrotic, characterized by the expression of CD20013. In addition to CD200, pro-fibrotic LEPR+ cells also express ACTA2 (αSMA), Tagln (SM22a), and FBLN2 13. Ablation of fibrotic LEPR+ cells leads to a reduction in peri-implant fibrous tissue, while transplantation of fibrotic LEPR+ cells is sufficient to induce the formation of peri-implant fibrous tissue.

Gremlin1 (GREM1) is a secreted antagonist of the bone morphogenetic protein (BMP)-2, -4, and -723. Upregulation of the BMP pathway was previously shown to increase peri-implant osteogenesis and mineralization24, and to increase recruitment of osteoprogenitors to the site of bone remodeling25. BMPs were also shown to impede fibrosis through inhibition of the transforming growth factor beta (TGFβ) pathways26. While the BMP pathway has been shown to contribute toward peri-implant bone formation, its role, particularly the role of the BMP inhibitor GREM1, in the formation of peri-implant fibrotic tissue and its correlation to the fibrotic LEPR+ cells remains to be elucidated.

Here, we show that GREM1 is prominently expressed by the majority of LEPR⁺ cells within peri-implant fibrotic tissue from both patients undergoing revision surgery and our murine model of peri-implant fibrosis. These GREM1⁺ LEPR⁺ cells co-express classical fibrotic markers (Col3a1, S100a4, CD200) as well as osteogenic markers (OSX, BGLAP), suggesting a diverted differentiation trajectory from osteogenesis toward fibrosis. Functional studies reveal that GREM1 expression in LEPR⁺ cells is required for the development of peri-implant fibrosis, as conditional deletion of Grem1 results in a marked reduction in fibrotic tissue and a concomitant increase in peri-implant bone formation. Mechanistically, Grem1 deletion leads to robust upregulation of both BMP and WNT signaling pathways, redirecting LEPR⁺ cells away from a fibrogenic fate and toward osteogenic differentiation. This pathway is mechanistically distinct from our previously described ADGRF5-mediated signaling, which we showed reduces fibrosis and enhances osteogenesis but does not influence BMP or WNT activation. Notably, pharmacologic inhibition of GREM1 with a neutralizing antibody phenocopies the genetic deletion, further supporting the translational potential of targeting GREM1. Together, these findings identify GREM1 as a novel, mechanistically independent from ADGRF5 signaling, and therapeutically actionable target for preventing and reversing peri-implant fibrosis.

Results
GREM1 is robustly expressed by LEPR+ cells in human and murine peri-implant fibrotic tissue
We hypothesized that the BMP pathway played a crucial part in peri-implant bone and fibrous tissue formation. To interrogate BMP-pathway–associated transcriptional changes under regenerative versus fibrotic conditions, we employed a murine tibial implantation system that generates either stable osseous integration27 or mechanically induced fibrotic encapsulation. Stable press-fit implantation reproducibly supported bone integration, whereas implantation into an overdrilled tibial canal introduced micromotion and promoted fibrous interface formation (referred to subsequently as fibrous-integrated surgery). LepR-lineage cells were isolated from both conditions for comparative transcriptomic analysis (Supplementary Figure 1a–b). Among differentially expressed genes, Grem1, a BMP-inhibitor, emerged as a candidate effector gene of interest in regulating the fibrotic activity of LepR-lineage cells in peri-implant fibrotic tissue.

To validate the transcriptomic findings at the protein level, we examined GREM1 expression in interfacial tissue collected from patients undergoing revision arthroplasty for aseptic loosening (Figure 1a–e, Supplementary Figure 2). Clinical diagnosis was supported by radiographic evidence of peri-implant lucency and intraoperative confirmation of mechanical instability. Histologic evaluation of the interface demonstrated a predominance of elongated stromal cells co-expressing LEPR and GREM1 (Figure 1b-d, Supplementary Figure 2) Notably, LEPR⁺ cells within fibrotic tissue displayed robust GREM1 expression, whereas LEPR⁺ cells in adjacent trabecular bone showed minimal GREM1 signal (Figure 1e, Supplementary Figure 2).

The presence of LEPR+ GREM1+ cells in human peri-implant fibrotic tissue led us to question whether LepR-tdTomato+ GREM1+ cells similarly comprised the peri-implant fibrotic tissue in a murine model. Similar to the human peri-implant fibrotic tissue, murine peri-implant fibrotic tissue contained LepR-tdTomato+ spindle-shaped fibroblasts that expressed GREM1 (Figure 1f-j, Supplementary Figure 1c, Supplementary Figure 3). Furthermore, similar to our observations in human fibrous tissue and normal bone, a significantly higher percentage of LepR-tdTomato+ cells in the peri-implant fibrotic tissue expressed GREM1 compared to LepR-tdTomato+ cells in normal peri-implant bone that underwent osseous integration (Figure 1k, Supplementary Figure 1c, Supplementary Figure 3). There was no statistically significant difference in the total percentage of LepR-tdTomato+ cells in the peri-implant fibrous tissue and the peri-implant bone between the inducible CreERT and the constitutionally active Cre (Supplementary Figure 3c-d). Taken together, under homeostatic conditions, LepR-lineage cells did not express GREM1, consistent with a previous study showing that Grem1-lineage cells represent less than 1% of LEPR+ cells21. Under pro-fibrotic conditions, both human and murine peri-implant LepR-lineage cells acquired expression of GREM1.

Periosteal cells minimally contribute to peri-implant fibrosis
To test the possibility that the periosteal stromal cells contributed toward peri-implant fibrotic tissue, we performed fibrous integration surgery on both Gli1-CreERT;tdTomato mice to track Gli1-lineage cells28, and Ctsk-Cre;mTmG to track Ctsk-lineage cells29.

Ctsk-lineage cells were found in the periosteum and endosteal osteoclasts as previously reported29 (Supplementary Figure 4a). However, Ctsk-lineage minimally contributed to the cells in the peri-implant fibrotic tissue (Supplementary Figure 4a), with only around 10 percent of the cells in the peri-implant fibrotic tissue being Ctsk-lineage (Supplementary Figure 4d). Gli1-lineage cells were found in both the periosteum and in the growth plate region, as previously reported (Supplementary Figure 4b)28. However, Gli1-lineage cells minimally contributed to peri-implant fibrotic tissue (Supplementary Figure 4b), with less than 10 percent of cells in the peri-implant fibrous being Gli1-lineage (Supplementary Figure 4d). Taken together, periosteal cells marked by Ctsk or Gli1 lineage reporters only made a minor contribution to peri-implant fibrous tissue, while LepR-lineage cells provided a significantly higher contribution to peri-implant fibrotic tissue.

LepR-lineage skeletal cells in the peri-implant fibrotic tissue express a fibrotic gene signature
The presence of LepR-tdTomato+ cells in the peri-implant fibrotic tissue led us to question whether these fibrotic LepR-tdTomato+ expressed fibrotic signature markers, such as COL3A130 and S100A413. To address this, we performed fibrous integration surgery on both LepR-CreERT;tdTomato;Col3a1-GFP to trace Col3a1-expressing LepR-tdTomato+ cells and LepR-CreERT;tdTomato;S100a4-GFP to trace S100a4-expressing LepR-tdTomato+ cells.

Using genetic reporters for COL3A1 and S100A4, LepR-tdTomato+ cells in the fibrotic tissue could be distinguished from other populations of LepR-tdTomato+ cells by their expression of COL3A1 (Supplementary Figure 5a-e) and S100A4 (Supplementary Figure 6a-e). In contrast, in the osseointegration surgery model, the LepR-tdTomato+ cells in the peri-implant osseous tissue expressed neither COL3A1 (Supplementary Figure 5f-j) nor S100a4 (Supplementary Figure 6f-j). A higher percentage of LepR-lineage cells was found in the peri-implant fibrotic tissue of mice that underwent fibrous integration surgery than in the peri-implant bone tissue of mice that underwent osseointegration surgery (Supplementary Figure 5k, 6k). A higher percentage of LepR-lineage cells in the peri-implant fibrotic tissue expressed COL3A1 and S100A4 than the corresponding LepR-lineage cells in the peri-implant osseous tissue in the osseointegrated press-fit model (Supplementary Figure 5l-m, 6l-m). Col3a1- and S100a4- expressing skeletal LepR-tdTomato+ cells were previously reported to represent the pro-fibrotic population in murine skeletal fibrosis models13,30. Taken together, LepR-lineage cells in the peri-implant fibrotic tissue expressed signature pro-fibrotic genes such as COL3A1 and S100A4, thus assuming a fibrogenic fate.

Fibrotic LepR-tdTomato+ cells express osteogenic markers OSX and BGLAP
We next sought to determine whether LepR-tdTomato+ cells in the peri-implant fibrotic tissue retain expression of osteogenic lineage markers, such as BGLAP31 and OSX32. We hypothesized that during the immediate postoperative period, marrow LepR-tdTomato+ cells expressed early osteogenic markers, such as OSX that would later evolve to include the expression of late osteogenic markers, such as BGLAP. Additionally, we hypothesized that the expression of osteogenic lineage markers by LepR-tdTomato+ cells follows a radial gradient from the endosteal bone, such that LepR-tdTomato+ cells closest to the endosteal bone progress from early to later stages of the osteogenic lineage at earlier time points than those located farther away. To address this question, we performed fibrous-integration surgery on LepR-CreERT;tdTomato;Osx-mCherry mice to trace OSX-expressing LepR-tdTomato+ cells, and LepR-CreERT;tdTomato;Bglap-GFP mice to trace BGLAP-expressing LepR-tdTomato+ cells (Figure 2). To further refine the spatial analysis of these cells and assess osteogenic lineage marker expression as a function of radial distance from the host endosteal bone, we divided the peri-implant fibrous tissue layer into three equal zones of increasing radial distance from the endosteal bone post-drilling. Zone one represented the one-third of the fibrous tissue closest to the host endosteal bone, zone two corresponded to the middle zone of the fibrous tissue, and zone three referred to the one-third of the fibrous tissue closest to the implant (Figure 2a-c).

At postoperative day 3, the peri-implant area consisted of early, disorganized peri-implant fibrotic tissue, which was mostly LepR-tdTomato- (Figure 2a). This early, disorganized peri-implant fibrotic tissue was OSX- (Figure 2a), BGLAP-GFP- (Figure 2g), and GREM1- (Figure 2m, Supplementary Figure 7). At the junction between trabecular bone and the early fibrotic tissue, the marrow space was filled with spindle-shaped LepR-tdTomato+ cells that are OSX+ (Figure 2a), BGLAP+ (Figure 2g), and GREM1+ (Figure 2m, Supplementary Figure 7). The proportion of OSX-expressing LepR-tdTomato+ cells peaked at postoperative day 7 in zones 2 and 3 (Figure 2b, d-f) and then declined at postoperative day 14 (Figure 2c, d-f). The proportion of LepR-tdTomato+ BGLAP+ and LepR-tdTomato+ GREM1+ cells continued to increase from postoperative day 3 to 14 in all zones (Figure 2g-r, Supplementary Figure 7). Taken together, early LepR-tdTomato+ cells in the peri-implant fibrous tissue expressed OSX. Over time, these LepR-tdTomato+ cells ceased expressing OSX and expressed BGLAP and GREM1.

Conditional deletion of Grem1 in LepR-lineage cells attenuated peri-implant fibrosis
To determine whether GREM1 expression in LepR-lineage cells influences peri-implant tissue composition, we conditionally deleted Grem1 in LepR-lineage cells (LepR-Cre;tdTomato;Grem1fl/fl, hereafter called LepR-Cre+;Grem1f/f mice). Twelve to sixteen week old LepR-Cre;tdTomato;Grem1fl/fl and LepR-Cre;tdTomato;Grem1+/+ (control, LepR-Cre+) underwent fibrous-integrated surgery and analyzed the interface two weeks following fibrous-integration surgery (Figure 3a). Micro-computed tomography analysis showed a statistically significant increase in peri-implant bone mass in LepR-Cre+;Grem1f/f mice compared to LepR-Cre+ control after fibrous-integrated surgery (Figure 3b-d). This finding was further supported by the histomorphometric analysis of the peri-implant tissue, demonstrating a significant reduction in peri-implant fibrosis in LepR-Cre+;Grem1f/f mice (Figure 3e-g, Supplementary Figure 8a-h). Micro-computed tomography and histomorphometric analyses of baseline, non-operated tibiae revealed no significant differences in bone mass between LepR-Cre+;Grem1f/f and LepR-Cre+ (Supplementary Figure 9). The frequency of Lin- LepR-tdTomato+ cells in LepR-Cre+;Grem1f/f mice was significantly decreased compared to LepR-Cre+ controls, as shown by flow cytometry and immunofluorescence (Figure 3h-j). In conjunction with the reduction in peri-implant fibrosis, we also observed a concomitant reduction in the number of fibrosis-associated LepR-tdTomato+ CD200+ cells post fibrous-integrated surgery (Figure 3k-l). The overall increase in the peri-implant bone mass and decrease in the peri-implant fibrous tissue in LepR-Cre+;Grem1f/f mice further manifested as an increase in its host bone-implant interface strength as compared to LepR-Cre+ mice (Figure 3m-n). Taken together, conditional deletion of Grem1 in LepR-lineage cells does not affect baseline bone quality but reduces the post-surgical frequency of LepR-lineage cells and consequently attenuates the formation of peri-implant fibrotic tissue.

GREM1 inhibition reduced cell proliferation and skewed cellular differentiation from a fibrogenic towards an osteogenic fate
Given the reduction in fibrotic tissue following Grem1 deletion, we next evaluated whether LepR-lineage cell expansion within the peri-implant niche was dependent on GREM1. To assess cumulative proliferation, we administered bromo-deoxyuridine (BrdU) to 12-16 week-old LepR-Cre;tdTomato;Grem1fl/fl and LepR-Cre;tdTomato;Grem1+/+ mice that underwent fibrous-integration surgery daily for 14 days, starting immediately after surgery (Figure 4a-d). Conditional knockout of Grem1 in LepR-tdTomato+ cells led to a significant decrease in LepR-tdTomato+ cell proliferation in the peri-implant area (Figure 4a-d, o).

To assess whether LepR-tdTomato+ cell-derived GREM1 direct the differentiation of LepR-tdTomato+ cells towards a fibrogenic fate, LepR-tdTomato⁺ cells isolated from from LepR-Cre+;Grem1f/f and LepR-Cre+ mice that underwent fibrous-integration surgeries were engrafted intramuscularly into recipient mice (Fig. 4e–n). While LepR-tdTomato+ cells from the LepR-Cre+ donors exclusively formed graft-derived fibrotic tissue after transplantation, LepR-tdTomato+ cells from LepR-Cre+;Grem1f/f donors formed bone nodules containing graft-derived osseous and adipose tissue after transplantation (Figure 4e-n). Micro-computed tomography analysis of the mineralized tissue demonstrated a significantly higher amount of mineralized tissue in graft-derived tissue from LepR-Cre+;Grem1f/f relative to the graft-derived tissue from LepR-Cre+ control donors (Figure 4p). Taken together, deletion of Grem1 in LepR-lineage cells resulted in a phenotypic switch in differentiation capacity due to cell intrinsic reprogramming.

Conditional deletion of Grem1 in LepR-lineage cells upregulated BMP and WNT Pathways
GREM1 is a member of the differential screening-selected gene aberrative in neuroblastoma (DAN) family of secreted BMP antagonist and selectively binds to BMP-2, -4, and -7 to inhibit BMP pathway signaling33. GREM1 also inhibits WNT signaling, and, accordingly, downregulation of GREM1 resulted in enhanced WNT signaling34. We hypothesized that inhibition of GREM1 in LepR-lineage cells resulted in upregulation of BMP and WNT Pathways, resulting in increased osteogenesis and downregulation of fibrosis.

To define the molecular consequences of Grem1 deletion, we performed transcriptomic analysis of LepR-tdTomato+ cells isolated from LepR-Cre+;Grem1f/f and LepR-Cre+ mice that underwent fibrous-integration surgery and subsequent gene set enrichment analysis (GSEA). Principal component analysis (PCA) established that LepR-tdTomato+ cells isolated from LepR-Cre+;Grem1f/f and LepR-Cre+ mice displayed distinct transcriptional profiles (Figure 5a). We identified enrichment of genes involved in BMP and WNT pathways in the LepR-tdTomato+ from LepR-Cre+;Grem1f/f (Figure 5b-c). BMP4 and BM7, but not BMP2, BMP6, and BMP9 were upregulated in the peri-implant tissue of LepR-Cre+;Grem1f/f mice compared to that of LepR-Cre+ mice (Supplementary Figure 10-12). Unlike GREM1, NOGGIN, another BMP-pathway inhibitor, was not significantly expressed by LepR-lineage cells in the peri-implant area (Supplementary Figure 12). Furthermore, phosphorylated SMAD (pSMAD) 1/5/8 and active β-catenin, markers of active BMP and WNT signaling respectively, were upregulated in the peri-implant tissue of LepR-Cre+;Grem1f/f mice compared to that of LepR-Cre+ mice (Figure 5d-i). By contrast, conditional deletion of Adgrf5 in LepR-tdTomato⁺ cells had no effect on Grem1 expression—and vice versa, Grem1 knockout did not alter Adgrf5 levels (Supplementary Figure 13)—and neither BMP nor WNT signaling was significantly perturbed in LepR-Cre⁺;Adgrf5f/f animals. These data demonstrate that GREM1, but not ADGRF5, serves as a critical brake on BMP/WNT-driven osteogenesis in the peri-implant niche.

In agreement with our submuscular engraftment result, conditional knockout of Grem1 in LepR-lineage cells resulted in upregulation of pro-osteogenic genes associated with BMP or WNT pathways (e.g. Alx335,36, Bmp737, Bmp438, Acvr2b39,40, Acvr1c41, Wnt2b42, Wnt643, Wnt444, Wnt10a43, Bglap45, and Bglap245, Efemp146,47, Ccn348 , Hoxd1349, Supplementary Figure 14a), upregulation of anti-fibrotic genes associated with BMP or WNT pathways (e.g. Bmp450, Bmp751, Wnt2b52, Wnt453, Wnt654, Klf1555, Pitx256,57, Efemp158, Fgfr459,60, Supplementary Figure 14b), downregulation of anti-osteogenic genes (e.g. Frzb61, Tns362, Nsd263, Panx364, Trpv465, Fgfr366, Osmr67, Lgr668, Icam269, Igf2r70, Supplementary Figure 14c), and downregulation of pro-fibrotic genes (Osmr71, Rasal172, Fzd973, Lgr674, Ets175, Flnb76, Hoxc877, Chst1178,79, Trpv465,80, Nid281, Igf2r82, Supplementary Figure 14d).

In concordance with our BRDU study, conditional knockout of Grem1 in LepR-lineage cells resulted in upregulation of anti-proliferation genes associated with BMP or WNT pathways (e.g. Podn83, Wnt484, Acvr1c85, Bmp486, Bmp787, Efemp158, Inmt88, Hepacam89, Eno390, Dpp491, Supplementary Figure 14e) and downregulation of pro-proliferation genes (e.g. Lgr692, Egfl793, Lef194, Panx395, Apln96, Notch497, Piezo198, Tert99, Stc1100, Dlk2101, Supplementary Figure 14f). Taken together, the deletion of Grem1 in LepR-lineage cells resulted in an upregulation of BMP and WNT pathway-associated osteogenic genes, and downregulation of pro-fibrotic and pro-proliferation genes and represent an orthogonal pathway from the previously reported Adgrf5 axis.

GREM1 blocking antibodies prevent peri-implant fibrosis and improve peri-implant osseointegration
Since conditional genetic deletion of Grem1 in LepR-lineage cells resulted in the reduction of peri-implant fibrosis, we hypothesized that administration of an anti-GREM1 blocking antibody would also prevent peri-implant fibrosis and promote peri-implant osteogenesis, therefore acting as prophylaxis against peri-implant fibrosis. Anti-GREM1 antibodies (previously shown to block GREM1 signaling102) were administered via intra-articular injections from postoperative day 0 to day 14 in LepR-Cre;tdTomato mice undergoing fibrous-integration surgery. (Figure 6a).

After a total of 14 days of treatment, μCT analysis showed an enhancement of peri-implant bone formation in anti-GREM1 treated mice as compared to mice treated with an isotype control (Figure 6b-d). The bone volume over total volume and the trabecular thickness in the peri-implant area were significantly higher relative to controls (Figure 6c-d, Supplementary Figure 8i-j). Histomorphometry analysis further showed a reduction in peri-implant fibrous tissue and an increase in peri-implant bone formation in the anti-GREM1 treated mice as compared to isotype controls (Figure 6e-g). Micro-computed tomography and histomorphometric analyses of the ipsilateral femur revealed no significant differences between mice treated with anti-GREM1 relative to mice receiving an isotype control (Supplementary Figure 15). Finally, flow cytometric analysis and immunofluorescence imaging demonstrated decreased numbers of both LepR-tdTomato+ and LepR-tdTomato+CD200+ cells in the peri-implant area of anti-GREM1 treated mice (Figure 6h-l). The overall increase in the peri-implant bone mass and decrease in the peri-implant fibrous tissue as seen in the microCT and histomorphometric analysis further manifests as an increase in bone-implant interface-strength, as compared to control mice (Figure 6m-n).

GREM1-blocking antibodies treat established peri-implant fibrosis
We then asked whether administration of anti-GREM1 can promote remodeling of established peri-implant fibrosis back into an osseus interface and, therefore, act as a treatment. To address this question, we administered anti-GREM1 starting from postoperative week 2 until postoperative week 4 to LepR-Cre;tdTomato that underwent fibrous-integrated surgery (Figure 7a).

Micro-computed tomography analysis showed an enhancement of peri-implant bone formation in anti-GREM1 treated mice as compared to mice treated with isotype control (Figure 7b-d). The bone volume over total volume and trabecular number in the peri-implant area were significantly higher relative to controls, while the trabecular spacing in the peri-implant area was significantly lower relative to controls (Figure 7c-d, f). Histomorphometric analysis further showed a significant reduction in peri-implant fibrous tissue and an increase in peri-implant bone formation in the anti-GREM1 treated mice as compared to isotype controls (Figure 7e, g-h, Supplementary Figure 9k-l). Micro-computed tomography and histomorphometric analyses of the ipsilateral femur revealed no significant differences between mice treated with anti-GREM1 relative to mice receiving an isotype control (Supplementary Figure 16). Flow cytometry analysis and immunofluorescence imaging demonstrated decreased numbers of both LepR-tdTomato+ and LepR-tdTomato+CD200+ cells in the peri-implant area of anti-GREM1 treated mice (Figure 7h-m). The overall increase in the peri-implant bone mass and decrease in the peri-implant fibrous tissue, as seen in the microCT and histomorphometric analysis, further manifested as an increase in host bone-implant interface-strength as compared to control mice (Figure 7n-o). Taken together, these results provided proof-of-principle that inhibition of GREM1 is a promising strategy to not only prevent but also to treat established peri-implant fibrosis.

Methods
Institutional approvals
All procedures were carried out in accordance with applicable ethical and regulatory requirements. Animal experiments followed NIH guidelines for the Care and Use of Laboratory Animals and were approved by the Weill Cornell Medicine Subcommittee on Animal Care (IACUC protocol 2018-0001). Procurement and use of human specimens were performed under approval from the Hospital for Special Surgery Institutional Review Board (IRB protocol 2019-0571), which included HIPAA authorization, and all study conduct adhered to ethical principles consistent with the 2008 Declaration of Helsinki. The IRB permitted the use of de-identified residual clinical specimens under a waiver of informed consent; accordingly, written informed consent was not required.

Animals
Lepr-cre (Stock 008320), Rosa26-CAG-loxp-stop-loxp-tdTomato (Stock 007909), Rosa26-CAG-loxp-stop-loxp-iDTR (Stock 007900), S100a4-GFP (Stock 012893), NSG-EGFP (Stock 021937) mice were obtained from Jackson Laboratories. Col3a1-GFP (Stoc 010616-UCD) mice was obtained from Mutant Mouse Resource & Research Centers (MMRRC). LepR-CreERT mice were provided by Bo Zhou (Chinese Academy of Sciences, China). Grem1fl/fl mice were a gift from Aris Economides (Regeneron, USA). All mice were maintained on a C57BL/6 background and housed/handled in compliance with NIH guidelines for the Care and Use of Laboratory Animals under protocols approved by the Weill Cornell Medical College Subcommittee on Animal Care (IACUC). For antibody studies, mice were randomized to receive anti-GREM1 versus isotype control. Microcomputed tomography (μCT) analyses were performed with blinding to experimental condition. Unless otherwise indicated, CreER recombination was induced by daily intraperitoneal tamoxifen injections for three consecutive days administered two weeks before surgery. Experiments used 10–18-week-old mice, with a minimum of 3 animals per experiment. Both male and female animals were included and randomized when assigned to groups. Post hoc sex-based statistical comparisons were not performed due to low sample size; sex-disaggregated values are provided in the Source Data.

Animal welfare and euthanasia
Animals were housed in an AAALAC-accredited, specific pathogen–free facility under standard conditions with ad libitum access to food and water, along with environmental enrichment. Trained personnel assessed mice at least daily and more frequently after surgery; monitoring included body condition, weight trends, mobility/limb use, grooming, and surgical-site integrity. Peri-operative analgesia and supportive care were delivered per institutional veterinary recommendations and the approved IACUC protocol. Humane endpoints—including >15–20% weight loss, persistent distress, impaired ambulation, or wound complications—prompted veterinary evaluation and, when indicated, early euthanasia. Euthanasia was performed by CO₂ inhalation with a secondary physical method (e.g., cervical dislocation) to confirm death, consistent with institutional practice, AVMA guidance, and IACUC protocol 2018-0001.

Human specimens
Human specimen use was approved under the Hospital for Special Surgery IRB protocol 2019-0571, which included HIPAA authorization and conduct consistent with the 2008 Declaration of Helsinki. The IRB allowed use of de-identified residual clinical material under a waiver of informed consent; therefore, written informed consent was not required. Peri-implant fibrous interface tissue was collected from 9 patients (5 males and 4 females, 40–85 years old) undergoing revision arthroplasty 2–10 years after primary total hip or total knee replacement for aseptic loosening; specimens analyzed were not required for clinical management. Patients revised for septicemia were excluded using Musculoskeletal Infection Society criteria107. Aseptic loosening was confirmed by radiographic peri-implant lucency and intraoperative evidence of a mechanically loose implant together with peri-implant fibrotic tissue on operative assessment. Interface tissue was harvested by blunt scraping of fibrotic tissue adjacent to the implant from the proximal femur or acetabulum in total hip arthroplasty cases, and from the distal femur or proximal tibia in total knee arthroplasty cases. Harvested specimens comprised predominantly fibrous tissue with a small amount of underlying bone, enabling assessment of both bone-to-fibrous tissue and implant-to-fibrous tissue interfaces. Control “normal bone” samples were obtained from 4 patients (2 males and 2 females, 50–73 years old) undergoing primary total hip arthroplasty (greater trochanter region) or primary total knee arthroplasty (proximal tibia). Sex- or gender-based analyses were not performed due to limited sample size. Identifying patient information was removed at the time of procurement.

Fibrous integration surgery
Mice were anesthetized with isoflurane delivered by nose cone (2% isoflurane, 2 L/min) and surgery was performed under sterile technique. An ~8 mm medial parapatellar incision was made in the operative knee. The quadriceps fibers were released medially to permit lateral subluxation of the patella and exposure of the tibial plateau. The anterior cruciate ligament was released at its tibial insertion and both menisci were excised. Using a fine-tip burr, articular cartilage and the proximal epiphysis were removed down to the level of the posterior cruciate ligament insertion. To model an end-stage aseptic loosening phenotype—specifically, formation of a fibrous interface between implant and host bone—a 1.4 mm diameter × 2.0 mm length hole was created in the medullary canal. A medical-grade poly(methyl methacrylate) implant (Simplex P, Stryker, New Jersey, USA) was inserted until its top surface was flush with the proximal tibia. The implant articulating surface measured 2.0 mm (major axis) × 1.5 mm (minor axis) with a thickness of 0.2 mm; the stem measured 2.0 mm in length and 1.0 mm in diameter. Knee range of motion was verified before closure. The extensor mechanism and skin were closed in layers using resorbable sutures. Postoperative analgesia was provided with buprenorphine (0.05 mg/kg subcutaneously) for the first 48 hours. Mice were allowed full weight bearing and unrestricted activity from the day of surgery until sacrifice.

Osseointegration surgery
Osseointegration surgery used the same exposure and initial preparation as fibrous integration surgery, with the following changes. After cartilage and proximal epiphysis removal, a 10.9 mm diameter × 2.0 mm length hole was created in the medullary canal. A direct metal laser sintered implant composed of medical-grade Ti6Al4V alloy was press-fit into the prepared cavity until flush with the proximal tibial surface. The dimensions of the implant articulating surface were 2.0 mm for the major axis and 1.5 mm for the minor axis, with a 0.2 mm thickness. The stem was 2.0 mm in length and 1.0 mm in diameter. Closure was performed as described above. Buprenorphine (0.05 mg/kg subcutaneously) was administered for the first 48 hours postoperatively. Animals were permitted full weight bearing and no activity restriction from surgery to sacrifice.

Isolation of mesenchymal cells
The proximal tibia (2 mm from the most proximal surface) was processed using combined mechanical disruption and enzymatic digestion. Tissue was minced with sterile scissors and digested at 37°C with constant agitation for up to 45 minutes in digestion buffer containing collagenase P (1 mg/ml; Roche, cat 11213857001) and dispase II (2 mg/ml; Roche, cat 04942078001). The reaction was quenched by adding α-minimum essential medium (αMEM) supplemented with 2% serum, followed by centrifugation to pellet cells. After removing the supernatant, pellets were resuspended in DNase I (2 units/ml) and incubated for 10 minutes at 37°C. αMEM with 2% serum was added, samples were triturated thoroughly, filtered through a 70 μm nylon mesh, and centrifuged again. The final pellet was used for fluorescence-assisted cell sorting (FACS).

Fluorescence-assisted cell sorting (FACS)
Single-cell suspensions were washed twice in ice-cold FACS buffer consisting of 2% FBS and 1 mM EDTA in PBS. Cells were then blocked for 30 minutes at 4°C using blocking buffer (1:100 dilution; BD Bioscience 553142). Antibodies were diluted in Brilliant Stain Buffer (BD Bioscience, 563794), and samples were incubated with primary antibody mixtures for 40 minutes at 4°C in the dark. Cells were washed twice with FACS buffer, followed by additional washes, and resuspended in FACS buffer containing DAPI (4–6 diamidino-2-phenylindole; 1 μg/ml; BD Bioscience, 564907). Sorting/acquisition was performed on a Becton Dickinson Aria II equipped with 5 lasers (BD Bioscience). Initial compensation was set using compensation beads (Invitrogen, 01-3333-42), then refined using fluorescence-minus-one (FMO) controls, which were also used to define background for each fluorophore. Gates distinguishing positive and negative populations were established using internal FMO controls. For each analysis, ~1–2.5 million events were typically collected. Data were analyzed in FlowJo (version 10.6); calculations were performed in Excel, and plots were generated in GraphPad Prism. Gating strategies are provided in Supplementary Figure 19.

FACS antibodies
For mouse flow cytometry, antibodies included CD45 (clone 30-F11, BD Bioscience), CD31 (clone MEC13.3, BD Bioscience), and Ter119 (clone Ter119, BD Biosciences). Flow cytometry was performed using a FACS Aria II (BD Biosciences).

Intramuscular engraftment
Intramuscular transplantation was carried out in 4–8-week-old NSG-eGFP mice (Stock 021937). A ~1 mm anterolateral incision was created on the right hindlimb to expose the right quadriceps. The quadriceps muscle complex was gently dissected and a 2–3 mm intramuscular pouch was formed. A Surgifoam absorbable gelatin sponge (Ethicon, 1972) loaded with the sorted cell population of interest was placed into the pouch. The site was closed in two layers to ensure full encapsulation of the sponge: first the fascial layer and then the skin. Bone nodules were harvested 8 weeks after implantation.

Transplanted cells were derived from FACS-sorted LepR-tdTomato+ cells isolated from the proximal tibia of LepR-Cre+;Grem1f/f and LepR-Cre+ mice following fibrous integration surgery. To obtain 5,000–10,000 cells per recipient mouse, 5–10 proximal tibiae from operated donor mice were collected on postoperative day 14.

Mechanical testing
Bone–implant interface strength was assessed by pullout testing as described previously108. After tibial isolation, the distal tibia was embedded in polymethylmethacrylate. The proximal tibia was carefully dissected to enable a custom fixture to clamp the implant beneath its plateau. The implant long axis was aligned with the pullout direction, and testing was performed on an EnduraTEC ELF3200 system (Bose, EdenPrairie, Minnesota). Implants were extracted at 0.03 mm/sec until complete removal. The peak pullout load (N) was obtained from the resulting load–displacement curves.

Intra-articular administration of an GREM1-blocking antibody
Neutralizing anti-GREM1 was administered intra-articularly to LepR-Cre;tdTomato mice undergoing fibrous integration surgery; the antibody has been previously shown to neutralize GREM1 activity102. In the prevention paradigm, dosing was performed twice weekly from postoperative day 0 through day 14 (Figure 4a). For treatment of established peri-implant fibrosis, dosing was performed twice weekly from postoperative day 14 through day 28 (Figure 5a). Each injection delivered 10 μL of a 10 μg/mL solution of anti-GREM1 (LSBio, LS-C125371) or isotype control (Abcam, ab91353) diluted in PBS. Prior to injecting, residual unbound antibody was aspirated.

For intra-articular delivery, mice were anesthetized with isoflurane by nose cone (2% isoflurane, 2 L/min). The skin was prepared with diluted povidone–iodine and allowed to dry. With the knee held at ~45° flexion, a sterile 26-gauge needle attached to an empty 10 μL syringe (Hamilton, CAL7635) was inserted just lateral to the patellar tendon and just superior to the tibial joint line. Synovial fluid was aspirated to create space. While maintaining needle position intra-articularly, the empty syringe was replaced with a second 10 μL syringe containing antibody or isotype, and the solution was injected into the joint.

Sample preparation for cryosectioning
Freshly harvested tissues were fixed in 4% paraformaldehyde (PFA) at 4°C for 6 hours, washed in PBS, and decalcified in 0.5 M EDTA for 8 days. Samples were then transferred to infiltration solution (20% sucrose + 2% PVP) and rocked until they sank. Specimens were embedded in OCT containing 15% sucrose and stored at −80°C. Sections (15 μm thick) were cut using a Leica cryostat.

To verify that implant removal after EDTA decalcification did not artifactually disrupt peri-implant tissue architecture, we compared histomorphometric outcomes between specimens in which the implant was removed post-decalcification and specimens processed without decalcification with the implant retained (Supplementary Figure 8). No statistically significant differences were detected in peri-implant fibrosis or peri-implant bone between these approaches (Supplementary Figure 8).

Immunohistochemistry (IHC)
Frozen sections were brought to room temperature and thawed for 10 minutes, rehydrated in PBS for 20 minutes, and permeabilized with 0.3% Triton X-100 in PBS for 15 minutes. Sections were then blocked in 5% donkey serum (PBS) before incubation with primary antibodies diluted in blocking buffer (overnight, 4 °C). After PBS washes, fluorophore-conjugated secondary antibodies (1:400) were applied, followed by nuclear counterstaining with 4’,6-diamidino-2-phenylindole (DAPI) (300 nM) for 10 minutes and mounting in antifade medium (Life Technologies, P36970). Confocal imaging was performed on a Zeiss LSM 880 with Airyscan using ZEN 2.3 software. Isotype controls were included to ensure minimum background signal, and image processing/quantification was performed using Carl Zeiss Zen 2.3 SP1 FP3 (black, v14.0.18.201, Germany).

Primary antibodies for IHC
Primary antibodies used for immunofluorescence included murine LEPR (R&D System, AF497, 1:100 dilution), human LEPR (R&D system, MAB867), GREM1 (LSBio, LS-C125371, 1:100 dilution), murine CD200 (Abcam, ab33734, 1:100 dilution), mouse active-β-Catenin (Milipore Sigma, 05-665, clone 8E7, 1:100 dilution), phospho Smad1/Smad5/Smad8 (EMD Milipore, AB3848-I, 1:100 dilution), Noggin (R&D System, AF719, 1:100 dilution), BMP2 (Abcam, ab214821, 1:100 dilution), BMP4 (Abcam, ab39973, 1:100 dilution), BMP6 (R&D System, AF6325, 1:100 dilution), BMP7 (Bioss USA, bs-2242R, 1:100 dilution), BMP9 (R&D System, MAB3209, 1:100 dilution).

Histological quantification
H&E staining were performed following the recommended protocol by the kit manufacturer (Abcam, ab245880), and whole-slide images were analyzed in QuPath109 (version 0.4.4). For each section, a peri-implant region of interest was defined as a 200 µm band extending outward from the implant interface. A supervised random-trees pixel classifier was trained to segment bone, fibrous tissue, and marrow/fat using manual annotations across multiple representative slides until performance was visually consistent. The trained classifier was then applied to the ROI for each sample, and segmented classes were converted to annotations to compute area fractions for each tissue compartment.

μCT scanning and analysis
Murine bones were scanned using a Scanco µCT 35 (6 µm isotropic voxel size; 55 kVp; 0.145 mA; 600 ms integration; 0.36° step over 180°, SCANCO Medical, Bassersdorf, Switzerland) while submerged in PBS108. Reconstructions were segmented to delineate implant, mineralized bone, and the radiolucent interfacial region. A peri-implant volume of interest was defined as a 200 µm shell extending outward from the segmented implant surface (Supplementary Figure 18). Standard trabecular metrics were computed in Scanco software following published guidelines outlined by Bouxsein et al., 2010110, including BV/TV and trabecular microarchitecture (trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and mean trabecular number (Tb.N)). Trabecular thickness, mean trabecular separation, and mean trabecular number were derived using sphere-fitting approaches implemented by the vendor. For trabecular thickness, spheres were fitted to the object, and for separation, spheres were fitted to the background111. Analyses were performed blinded to genotype/treatment.

Bulk RNA sequencing
LEPR+ cells were FACS-purified from the murine peri-implant region at postoperative day 14. RNA was isolated using the RNeasy Micro Kit (Qiagen, Germantown, MD, USA) and used for library preparation with the Illumina TruSeq RNA Sample Preparation workflow. Libraries were sequenced to ~15–25 million single-end reads per sample using HiSeq2500 with 50-bp single-end reads. Adapters and low-quality bases were removed with fastp112., and reads were aligned to the mouse reference genome (mm10) with STAR using Gencode v27 annotations for exon counting113.. Differential expression was assessed in R using edgeR (quasi-likelihood framework). Differential expression analysis was performed using the DESeq2 package and visualized in R using Shiny-driven visualization platform (RNAseq DRAMA, https://gitlab.com/hssgenomics/Shiny).

Statistical analyses
Data are reported as mean ± standard deviation (S.D.) or standard error of the mean (S.D.), as specified in each legend. Normality was assessed using the Shapiro–Wilk test where applicable. Two-group comparisons were performed using unpaired, two-tailed Student’s t-tests for normally distributed data or Mann–Whitney tests otherwise. For ≥3 groups, one-way ANOVA with Tukey’s multiple-comparisons test was used when assumptions were met; otherwise, Kruskal–Wallis testing followed by Dunn’s correction was applied. Analyses were performed in GraphPad PRISM software (v8.4.3, La Jolla, CA). P < 0.05 was considered significant.

Data Availability
The main data supporting the results in this study are available in the paper and supplementary information. The raw and analyzed data generated during the study are available for research purposes from the corresponding author within reasonable request. Transcriptomic data from bulk RNA-seq has been deposited at the Gene Expression Omnibus (GEO) under accession numbers GSE313854 and GSE227869. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Suhardi and colleagues addresses the problem of fibrous encapsulation, e.g., where an implant loosens and becomes encased in a fibrous tissue envelope rather than bone. The causes of fibrous encapsulation range from biological to physical e.g., excessive/early loading; consequently, there is clinical value in understanding at least part of the etiological basis for fibrous encapsulation.\nThe authors show evidence that LepR+ cells contribute to peri-implant fibrotic tissue, and that Grem1 is expressed in the same location. They then provide data showing that knockout of Grem1 leads to enhanced WNT and BMP signaling and associated downstream events including osteogenic differentiation. The daily delivery of an anti-GREM1 antibody appears to reduce the formation of fibrous tissue even in a femoral implant that is placed into an oversized osteotomy. This is an important finding because it suggests that a biological factor can override the role of physical forces/excessive micromotion that typically results in fibrous encapsulation. Fibrous encapsulation can also be reversed by delivery of the anti-GREM1 antibody, leading the authors to postulate that a anti-GREM1-mediated therapy may be of value in reducing the incidence of fibrous encapsulation.\nThis proposed therapeutic strategy, however, does not take into account the fact that implant loosening is a gradual process and its onset varies depending on factors such as implant type, surgical technique, etc. To employ an anti-GREM1 antibody strategy as a preventative would require extensive safety data demonstrating that manipulation of BMP and WNT pathways holds no short- or long-term safety issues. This important point is not discussed.\nNonetheless, the paper makes some important strides in understanding the molecular mechanisms of fibrous encapsulation around femoral (not dental) implants and in this limited application, presents an important but potentially incremental advance in the field of orthopedics.\nMajor technical limitations\n1. In patients, early fibrous encapsulation is often associated with early/excessive loading of the implant. The effect of excessive micromotion is thought to produce strains and stresses in the peri-implant bone that cause it to break down and the bone tissue is then replaced by fibrous tissue. This sequence of events is not replicated in the murine fibrous integration surgery. Instead, fibrous encapsulation is ensured because the implant is undersized relative to the diameter of the osteotomy (1.0mm in diameter for the implant and 1.4mm in diameter for the osteotomy). Consequently, the implant is “floating” in the hole.\n2. The implant used in the fibrous encapsulation surgeries is unlikely to w undergo osseointegration because it is fabricated from PMMA and not titanium. There are no threads on the implant used; consequently, it is impossible to ascertain whether we’re looking at the actual tissue-implant interface, as those shown in Fig. 3e. If there was fibrous tissue in zone 3, this could easily be lost during tissue processing.\n3. Fig. 3: “we conditionally deleted Grem1 in LepR-lineage cells); when was the deletion performed relative to the fibrous integration surgery? This information is not presented in the Methods section. If, for example, Grem1 was inactivated prior to surgery this could explain the significant increase in BV/TV, Tb.N, Tb.S, etc., unrelated to the surgical procedure. Since there is no mention of when Grem1 is inactivated it is not possible to answer this question.\n4. Fig. 4: the authors write that they delivered BrdU “for 14 days, starting immediately after surgery”. This is confusing because it suggests that BrdU was delivered repeatedly. Perhaps they mean that they delivered BrdU once, and traced the BrdU+ cells for 14 days? In any case, the extent of red positive cells is striking. If these represented cells that were proliferating immediately after surgery then there is apparently no turnover e.g., apoptosis and instead widespread proliferation. I have trouble reconciling that interpretation with most existing data that suggest extensive necrosis and apoptosis immediately after surgery, with proliferation only beginning ~72h after surgery.\n5. The authors begin by mentioning the number of dental implants and orthopedic placed, and the incidence/prevalence of failure due to aseptic loosening. This gives the false impression that all implants are somehow equal. While implants placed in the oral cavity and those placed in the appendicular skeleton can both undergo fibrous encapsulation and failure, comparative studies have clearly demonstrated that the molecular and cellular mechanisms of soft and hard tissue integration in the oral cavity and in the long bones are distinct and separable processes. Consequently, an investigation of the etiological basis for loosening of femoral implants should limit the rationale for undertaking the study to the type of implants actually examined e.g., orthopedic implants.\nMinor technical limitations\n1. Fig. 2 shows that bone ingrowth occurs around the implant between post-op day 3-14; what might the interface look like if it was examined on post-op day 30?\n2. The magnification of Fig. 2m, lower panel does not appear to be correct. The image shows the entire field from the bone to the implant surface. Also¬, there seems to be very little overlap between Grem1 and LepR. This does not align with the quantification data, however, which indicates a significance of 0.0001.\n3. Fig. 3, panel b: the pseudo-colored image superimposed on the micro-CT data obscures what looks like mineralized tissue in the region that is supposedly fibrous tissue.\n4. In Fig. 3, panel e: a histologic stain that distinguishes bone from fibrous tissue should be used rather than H&E.\n5. In Fig. 3 the authors show expression of CD200 and indicate in the Introduction that it is a marker of skeletal progenitor cells (line 49) then later (line 54) state that CD200 is a marker of fibrosis. Other data indicate it is an immune regulatory molecule. Given the multitude of functions assigned, this does not seem to be a good indicator of fibrosis. I would suggest the use of fibronectin or another well-defined fibrosis marker.\n6. Figure 4: the authors refer to the transplantation of LepR cells into a muscle pouch as an “osseous organoid”. Organoids are generally agreed to be three-dimensional cellular structures that are derived from tissue-specific stem cells and grown in vitro. What the authors show instead is a bone nodule. The histology shown is not indicative of bone, cartilage and fat staining. Positive controls should be included so readers understand the histologic appearance of the tissues.\n7. Figure 6: please clarify that the anti-GREM1 antibodies were delivered daily for 2 weeks. Please include data demonstrating that the anti-GREM1 used here is a function-blocking antibody.", "reviewer_2": "It is very exciting to see this kind of manuscript in the field of implantology that was dominated by biomechanics, engineering, and cellular biology. The vast majority of existing papers are descriptive in nature.\nThis study has many strengths such as identification of therapeutic targets in the human pathology implant loosening membranes, development of animal models with intact and mutant Grem1 function, neutralizing antibody treatments, and comparative transcriptomic analysis. The mouse animal model is rigorously analyzed by multiple IHC and quantitative microCT.\nThis study seems to be the first comprehensive mechanistic study that entailed the verification of Grem1 expression in LEPR+ cells in human tissue, and then replication aseptic loosening in clinically relevant wild type and mutant mice, therapeutic intervention using Grem1 neutralizing antibody, and further screening experiments for future experiments. All these features are exceptional strengths because literature on peri-implant fibrosis and loosening are at the cellular descriptive level. Loss of Grem1 function in the context of peri-implant osseous-integration changes the proliferation and tissue differentiation of the LEPR+ cells. To this end, one additional ‘Figure’ (Diagram or Cartoon) that explains the mechanism of peri-implant fibrosis, osseous integration, and proliferation of peri-implant cells would help readers understand the key concepts and therapeutic intervention. Obviously, this would not require any additional experiments. In the discussion section, please add or clarify whether Grem1 is a secreted protein that affects adjacent cells for differentiation into fibrous vs osteogenic cells (paracrine) or alters intracellular signaling for fibrous/osteogenic phenotypic changes (autocrine). These possible pathways could be incorporated into the Diagram. If the tissue is available for analyses, were there any difference in the number of osteoclasts? Excessive osteoclastic bone resorption is another mechanism of aseptic loosening. If not, please add sentences in the discussion that the focus of this study is about the diversion of the peri-implant skeletal stem cells into fibrous tissues and osteogenic tissues. Please also mention that osteoclastic bone resorption around the implant is triggered and aggravated by wear particles. In the animal model that was used in this experiment, wear-particle related inflammation and osteoclast formation was not relevant.\nOverall, the study is outstanding by unraveling the molecular mechanism of peri-implant fibrosis by highly mechanistic experiments using genetic loss-of-function and neutralizing antibody in a clinically relevant animal model.", "reviewer_3": "The subject of the manuscript is of significant interest to the orthopaedic and dental fields since loosening of endoprostheses and dental implants is a great medical problem. Authors based their entire hypothesis on the idea that the major problem outside the implant is the development of fibrous tissue associated with leptin receptor positive cells which express gremlin 1, one of well known BMP antagonists. In the provided evidence authors show four human tissues collected during surgical interventions following implant loosening, and various genetically modified mice following bone implants. Although authors provide a robust set of data, almost the entire evidence is based on dark field fluorescence microscopy without a real evidence into the relationship between bone, fibrous tissue and implant surfaces. Namely, in Figures 3 and 6 microCT images and related bright field histology at low magnification the presence and separation of fibrous tissue and surrounding bone are not well visualized. In Figure 4b the fibrous area separating the bone from implant is outlined as red rastered area; however, in the Figure 4b Leprcre/+ mice authors use red rastered area to indicate a large area of fibrous tissue, under which at the implant bottom the cancellous bone is protruding beneath the red, so called fibrous tissue. A similar impression exists in Figure 6b, when using a Grem1 antibody to decrease the fibrous area. Similarly, in Figure 4e the histology does not help in better separation of fibrous tissue and bone. Since fibrous tissue on microCT images is black, authors should have, instead of introducing red rastered self-drawing area, simply left the original image with fibrous tissue in originally black appearance. Since the entire further analyses depend on the original well visible images, all other analyses are not convincing since all presentations are in dark field fluorescence, witch completely misses associated bright field images to provide convincing evidence that analyzed cells and molecules are within the fibrous tissue and related markers stain cells, or areas of extracellular matrix outside the cells in relation to the implant and bone surfaces.\nThe mechanism indicating enrichment of the BMP pathway via analysis of relevant genes involved is not specific, since it is well known that different BMPs have different relationship with BMP inhibitors. It is unlikely that only Gremlin 1 exists in the fibrous area which is in tight contact with the surrounding bone in which the prominent antagonist is not Gremlin, but Noggin. Depending on the presence of BMP2, -4, -6, -7, and -9 (maybe 12-14), Noggin inhibits BMP2, -4 and -7, while BMP6 and BMP9 escape inhibition by Noggin due to one aminoacid difference (lysin) in the position 60 of the N terminal part of the molecule. Authors mention that Gremlin inhibits BMP2 and -7, but did not mention its effect on remaining BMPs, neither analyzed the fibrous tissue for specific BMPs and their inhibitors to make their mechanistic proposal fully reliable. It might be that Noggin is present as well in the fibrous tissue, including BMP6 or BMP9, which makes then the enrichment of BMP pathway analysis less convincing.\nSpecific issues:\n1. In Abstract authors claim that 18 million dental implants and 2 million total joint implants are placed annually.\n2. However, in Introduction they suddenly claim 18 million dental implants and 1.3 million total joint implants.\n3. In Figure 1a and 1f yellow boxes are too small to provide any information. In 1g scale bar in the text of 250 micrometers is 500 micrometers. In 1e and 1k a similar description is repeated, while e refers to human and k to mouse.\n4. In Figure 2 different graphs quantifying the colocalization refer to different numbers of samples and claim that they represent three independent experiments. This should be clarified, since it is not clear how many samples are from each of three experiments included in the analysis.\n5. Figure 4 presents in e and j data on bone organoids, which are of low imaging quality.\n6. Instead of staining bone and fibrous tissue with hematoxylin, the bone should have been presented by von Kossa mineral stain (black) which would eventually separate bone and fibrous tissue.\n7. Instead of BMP pathway gene analysis, which seems less relevant for this study, a structure function analysis of BMPs vs. Gremlin 1 would have been much more helpful to demonstrate inhibition of BMP6 as well, which might be present in adjacent bone, escaping the Noggin inhibition.\n8. The majority of small letters in figures throughout the manuscript are too small and not readable, see extended Figure 7 as an example.", "reviewer_4": "General comments: The MS by Suhardi et al reports that exuberant expression of GREM1 by Lepr+ cells drives fibrosis and the expense of osteogenesis at sites of implant placement into bone and that reducing GREM1 via local injection of neutralizing antibody reduces fibrosis and enhances osteogenesis. As peri implant fibrosis is a significant clinical problem and treatment with GREM1 neutralizing antibody is a simple clinical procedure, these findings may be of high impact. However, as presented the data raise concerns about the variable nature of the response to GREM1 antibody treatment in mice (see below for specifics) and that, coupled with the small number of human samples assayed for the presence of GREM1 (n=4 with a wide age range) at sites of peri implant fibrosis, raise concerns about the therapeutic potential of these findings. Additionally, the authors published a very similar finding in 2024 by targeting ADGRF5 in the same Lepr+ cell population but do not compare their new findings to these already published studies, raising concerns about the significance of targeting any one gene product as a therapeutic approach to peri-implant fibrosis and reducing the overall novelty of the current study.\nSpecific comments:\n1. Line 85: there seems to be a sentence missing about the identification of GREM1.\n2. Materials and methods: there is a description of isolation of mesenchymal cells from bone, but it is unclear if the cells retrieved include a heterogeneous population including periosteal cells, trabecular and cortical bone osteoblasts and bone marrow. As the number and types of Lepr+ cells would change as a function of age and health status in patients, it is unclear what this mixed population is a model of.\n2. Figures would benefit from inclusion of a representative histology section that allows the reader to orient to the immunolocalizations. It is hard to gauge how representative the areas focused on in the panels are in relation to the actual overall histology. This is especially important for the figures (2 and3) where comparisons are being made along the surface of the implant-tissue interfaces.\n3. Discussion:\na)The authors suggest that Lepr+ cells need to initiate osteogenesis to produce GREM1 and state that GREM1 expression blocks osteogenesis. It isn't clear from this model why GREM1 would be constantly on if osteogenesis is not occurring. No mention is made about what specifically regulates GREM1 expression in the Lepr+ cells. It is also unclear if all Lepr+ cells have equal ability to become osteoblasts and produce GREM1.\nb) No mention is made about how stable the rescue of osteogenesis is with injection of the GREM1 antibody. How does the antibody disrupt the feedback loop between osteoblast differentiation and GREM1 production and what happens when the antibody is no longer present as would be the case for treating patients?\nc) The authors state that both the BMP and Wnt pathways are enhanced when GREM1 is blocked but do not discuss which of these signals is required for the reinitiating of osteogenesis.\nd) The authors do not address how current findings with blocking GREM1 compare to those they reported in 2024, in the same animal model, of blocking ADGRF5. As the authors claimed anti ADGFR5 therapy was efficacious, it is incumbent upon them to show why additional therapy to block GREM1 is necessary and provides better results than blocking ADGFR5"}, "author_response": "9/2/2025\nRe: Manuscript NCOMMS-25-07110 “GREM1 acts in leptin receptor-expressing skeletal cells to\nmediate peri-implant fibrosis”\nPoint-by-point response\n(Reviewer comments are in red, our response is in black)\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Suhardi and colleagues addresses the problem of fibrous encapsulation,\ne.g., where an implant loosens and becomes encased in a fibrous tissue envelope rather than\nbone. The causes of fibrous encapsulation range from biological to physical e.g.,\nexcessive/early loading; consequently, there is clinical value in understanding at least part of the\netiological basis for fibrous encapsulation.\nThe authors show evidence that LepR+ cells contribute to peri-implant fibrotic tissue, and that\nGrem1 is expressed in the same location. They then provide data showing that knockout of\nGrem1 leads to enhanced WNT and BMP signaling and associated downstream events\nincluding osteogenic differentiation. The daily delivery of an anti-GREM1 antibody appears to\nreduce the formation of fibrous tissue even in a femoral implant that is placed into an oversized\nosteotomy. This is an important finding because it suggests that a biological factor can override\nthe role of physical forces/excessive micromotion that typically results in fibrous encapsulation.\nFibrous encapsulation can also be reversed by delivery of the anti-GREM1 antibody, leading the\nauthors to postulate that a anti-GREM1-mediated therapy may be of value in reducing the\nincidence of fibrous encapsulation.\nThis proposed therapeutic strategy, however, does not take into account the fact that implant\nloosening is a gradual process and its onset varies depending on factors such as implant type,\nsurgical technique, etc. To employ an anti-GREM1 antibody strategy as a preventative would\nrequire extensive safety data demonstrating that manipulation of BMP and WNT pathways holds\nno short- or long-term safety issues. This important point is not discussed.\nNonetheless, the paper makes some important strides in understanding the molecular\nmechanisms of fibrous encapsulation around femoral (not dental) implants and in this limited\napplication, presents an important but potentially incremental advance in the field of\northopedics.\nWe thank the reviewer for their thoughtful and encouraging comments on our manuscript. We\nappreciate the recognition of the significance of our findings in uncovering a molecular\nmechanism underlying fibrous encapsulation and the potential therapeutic relevance of GREM1\ninhibition.\nWe fully acknowledge that our current model does not capture the full temporal dynamics of\nimplant loosening, which is indeed a gradual and multifactorial process influenced by implant\ntype, surgical technique, mechanical loading, and host biology. Our model was intentionally\ndesigned to replicate the end-stage phenotype of aseptic loosening—specifically, the\npresence of a fibrous interface between the implant and host bone—rather than to model the\nentire progression of implant failure over time. This reductionist approach allows us to dissect\nthe molecular and cellular mechanisms that sustain fibrous encapsulation once it is established.\nWe have added a discussion of this point to both the methods and discussion\nsections(highlighted in yellow).\nRegarding the therapeutic potential of anti-GREM1 treatment, we agree with the reviewer that\nany prophylactic or long-term application would necessitate extensive safety evaluation,\nparticularly given the roles of BMP and Wnt signaling in diverse physiological processes.\nNeutralizing antibodies against GREM1 have recently been shown to have good safety profile in\na phase one clinical trial (Rivera IR, et al. Cancer Res, 2024, 5977; Sarker D, et al. Cancer Res,\n2023, CT094). Furthermore, analysis of the contralateral limbs in mice receiving intra-articular\nanti-GREM1 antibody injections showed no statistically significant differences in key bone\nparameters—including bone volume/total volume of cancellous bone in distal metaphysis and\ncortical thickness—based on both microCT and histological assessments (Extended Figure 15\nand 16). These findings suggest that local inhibition of GREM1 does not adversely impact\nsystemic or adjacent skeletal integrity, supporting the feasibility of a localized therapeutic\nstrategy. We have now included a discussion of these important safety considerations in the\nrevised manuscript (highlighted in yellow).\nGiven that over one million joint replacements are performed annually, with aseptic loosening\nbeing a leading cause of long-term failure, we believe that identifying Grem1 as a molecular\ndriver of fibrous encapsulation represents an important step towards non-surgical therapies. The\ndemonstration that targeting GREM1 can restore bone-implant integration, even in mechanically\nunfavorable conditions, supports its potential as a druggable target for improving implant\nlongevity.\nWe have revised the manuscript to explicitly address these points and to clarify both the scope\nand the limitations of our current findings (highlighted in yellow).\n1. In patients, early fibrous encapsulation is often associated with early/excessive loading of the\nimplant. The effect of excessive micromotion is thought to produce strains and stresses in the\nperi-implant bone that cause it to break down and the bone tissue is then replaced by fibrous\ntissue. This sequence of events is not replicated in the murine fibrous integration surgery.\nInstead, fibrous encapsulation is ensured because the implant is undersized relative to the\ndiameter of the osteotomy (1.0mm in diameter for the implant and 1.4mm in diameter for the\nosteotomy). Consequently, the implant is “floating” in the hole.\nWe thank the reviewer for this important observation. We fully acknowledge that, in patients,\nearly fibrous encapsulation is often precipitated by excessive micromotion and abnormal\nmechanical loading, which lead to elevated peri-implant strain, bone resorption, and ultimately\nfibrous tissue formation. While our murine model does not replicate the dynamic progression of\nmicromotion-induced fibrous encapsulation, it was purposefully designed to recapitulate the\nend-stage phenotype observed in clinical cases of aseptic loosening—namely, the formation of\na fibrous membrane between the implant and host bone. We recognize this limitation and have\nnow explicitly addressed it in the revised Discussion section to clarify the scope and\ntranslational relevance of our model (highlighted in yellow).\n2. The implant used in the fibrous encapsulation surgeries is unlikely to undergo\nosseointegration because it is fabricated from PMMA and not titanium. There are no threads on the\nimplant used; consequently, it is impossible to ascertain whether we’re looking at the actual\ntissue-implant interface, as those shown in Fig. 3e. If there was fibrous tissue in zone 3, this\ncould easily be lost during tissue processing.\nWe thank the reviewer for raising this important point. We agree that implant material and\ngeometry can influence both tissue integration and the interpretation of histological sections. In\nour model, we used smooth, non-threaded PMMA implants, which are not intended to\nosseointegrate but rather to consistently induce a fibrous interface. The absence of threads\ninherently limits mechanical interlocking with the surrounding tissue, which may help minimize\nthe risk of disrupting the interface tissue during implant removal, as the implant can slide\nout more easily without exerting traction on the adjacent structures.\nTo further address this concern, we performed a complementary set of experiments in which\nsamples were processed either with the PMMA implant left in place (via plastic embedding) or\nwith the implant removed prior to sectioning (Extended Figure 8). As described in the Methods\nsection, quantitative analysis revealed no statistically significant differences in the\npercentage of fibrous tissue or bone between these two approaches. These findings indicate\nthat our standard histological preparation method preserves the peri-implant tissue interface and\nsupports the accuracy of our assessments.\nWe have clarified these points in the revised Methods sections (highlighted in yellow).\n3. Fig. 3: “we conditionally deleted Grem1 in LepR-lineage cells); when was the deletion\nperformed relative to the fibrous integration surgery? This information is not presented in the\nMethods section. If, for example, Grem1 was inactivated prior to surgery this could explain the\nsignificant increase in BV/TV, Tb.N, Tb.S, etc., unrelated to the surgical procedure. Since there\nis no mention of when Grem1 is inactivated it is not possible to answer this question.\nTo clarify the timing of Grem1 deletion relative to the fibrous integration surgery, we\nadministered tamoxifen two weeks prior to surgery to induce recombination and inactivate\nGrem1 in LEPR-lineage cells. Tamoxifen was given once daily for three consecutive days,\nfollowed by an 11-day washout period before surgery. This timeline was designed to ensure\nefficient Cre-mediated recombination and avoid confounding effects from acute tamoxifen\nadministration. We have added this information in the methods section (highlighted in yellow).\nTo determine whether Grem1 deletion alone—independent of surgery—might affect baseline\nbone parameters, we analyzed the tibia in both Grem1-conditional knockout and control mice.\nQuantitative microCT and histological assessment revealed no significant differences in bone\nvolume fraction (BV/TV), trabecular number (Tb.N), or trabecular thickness (Tb.Th) between\ngroups (Extended Figure 9). These findings suggest that the observed increase in peri-implant\nbone formation is not attributable to global skeletal changes resulting from Grem1 deletion but\nrather reflects a localized response to surgical implantation.\nThis information has now been added to the revised Methods and Results sections (highlighted\nin yellow).\n4. Fig. 4: the authors write that they delivered BrdU “for 14 days, starting immediately after\nsurgery”. This is confusing because it suggests that BrdU was delivered repeatedly. Perhaps\nthey mean that they delivered BrdU once, and traced the BrdU+ cells for 14 days? In any case,\nthe extent of red positive cells is striking. If these represented cells that were proliferating\nimmediately after surgery then there is apparently no turnover e.g., apoptosis and instead\nwidespread proliferation. I have trouble reconciling that interpretation with most existing data\nthat suggest extensive necrosis and apoptosis immediately after surgery, with proliferation only\nbeginning ~72h after surgery.\nWe thank the reviewer for this thoughtful comment and the opportunity to clarify our\nexperimental design. To assess the cumulative proliferative capacity of LEPR-lineage cells in\nLepR-CreERT2+;Grem1fl/fl and cre-only controls, we administered intraperitoneal BrdU once\ndaily for 14 consecutive days, beginning immediately after the fibrous integration surgery. This\nrepeated BrdU dosing strategy was chosen to label all cells entering S-phase during this post-\nsurgical window, thereby capturing the aggregate proliferation response to implantation over\ntime. We have now clarified this protocol in the results section (highlighted in yellow).\n5. The authors begin by mentioning the number of dental implants and orthopedic placed, and\nthe incidence/prevalence of failure due to aseptic loosening. This gives the false impression that\nall implants are somehow equal. While implants placed in the oral cavity and those placed in the\nappendicular skeleton can both undergo fibrous encapsulation and failure, comparative studies\nhave clearly demonstrated that the molecular and cellular mechanisms of soft and hard tissue\nintegration in the oral cavity and in the long bones are distinct and separable processes.\nConsequently, an investigation of the etiological basis for loosening of femoral implants should\nlimit the rationale for undertaking the study to the type of implants actually examined e.g.,\northopedic implants.\nWe thank the reviewer for this important observation. We fully agree that the biological\nenvironments surrounding dental and orthopedic implants are fundamentally distinct, and that\nthe molecular and cellular mechanisms governing soft and hard tissue integration in the oral\ncavity versus the appendicular skeleton are not equivalent. While fibrous encapsulation is a\nshared pathological outcome, the etiological context differs substantially between these settings.\nWe have revised the Introduction to focus specifically on aseptic loosening in orthopedic\nimplants, particularly those placed in the femur. We have removed general references to dental\nimplants to ensure that the rationale for our study remains clearly grounded in the context of\northopedic implant failure.\nMinor technical limitations\n1. Fig. 2 shows that bone ingrowth occurs around the implant between post-op day 3-14; what\nmight the interface look like if it was examined on post-op day 30?\nOur previous findings (Suhardi et al., Nat. Biomed. Eng, 2024, 8:1285-1307) demonstrated that\nthe composition of the peri-implant interface—specifically the percentage of bone and fibrous\ntissue—remains relatively stable after day 14 in the absence of further mechanical perturbation.\nWe anticipate that the redirection of pro-fibrotic LEPR⁺ cells toward the osteogenic lineage by\nanti-GREM1 antibody results in the establishment of bone at the implant interface, which in turn\nreduces or eliminates micromotion, which is a primary stimulus for peri-implant fibrosis. We\nhave added this explanation in the discussion (highlighted in yellow).\n2. The magnification of Fig. 2m, lower panel does not appear to be correct. The image shows\nthe entire field from the bone to the implant surface. Also¬, there seems to be very little overlap\nbetween Grem1 and LepR. This does not align with the quantification data, however, which\nindicates a significance of 0.0001.\nThank you for pointing this out. We have updated Figure 2m so that the lower-panel inset shows\na correctly magnified view of the peri-implant interface. To clarify the apparent overlap between\nGREM1 and LEPR, we have also added separate single-channel images for GREM1 and LEPR\nalongside the merged overlay in Extended Figure 7.\n3. Fig. 3, panel b: the pseudo-colored image superimposed on the micro-CT data obscures what\nlooks like mineralized tissue in the region that is supposedly fibrous tissue.\nTo improve the visibility of the underlying micro-CT data in Fig. 3b, we have adjusted the\npseudo-colored overlays to be more transparent. This modification allows clearer visualization\nof mineralized tissue adjacent to the implant. The fibrous tissue is represented as the\nradiolucent gap between the implant (marked by a yellow rastered area) and the host bone\n(marked by a red rastered area).\n4. In Fig. 3, panel e: a histologic stain that distinguishes bone from fibrous tissue should be used\nrather than H&E.\nWe thank the reviewer for this helpful suggestion. In response, we have added Movat’s\npentachrome staining to enhance histological contrast between bone, fibrous tissue, and other\nmatrix components in the peri-implant region (Extended Figure 8). This stain allows for\nadditional visual differentiation of tissue types in addition to H&E.\nImportantly, quantitative analysis of peri-implant bone and fibrous tissue composition using\nMovat’s pentachrome staining yielded results consistent with those obtained from H&E-stained\nsections, supporting the robustness of our original findings. We have incorporated these\nadditional histological images and corresponding quantification into the revised manuscript.\n5. In Fig. 3 the authors show expression of CD200 and indicate in the Introduction that it is a\nmarker of skeletal progenitor cells (line 49) the later (line 54) state that CD200 is a marker of\nfibrosis. Other data indicate it is an immune regulatory molecule. Given the multitude of\nfunctions assigned, this does not seem to be a good indicator of fibrosis. I would suggest\nthe use of fibronectin or another well-defined fibrosis marker.\nThank you for your comment. Based on the reviewer suggestion, we have added Col3a1 and\nS100a4 (also known as fibroblast-specific protein or Fsp1) as fibrotic markers (Extended figure\n5 and 6). As shown in Extended Figure 5 and 6, the vast majority of LEPR⁺ cells within the\nperi-implant fibrotic niche co-express Col3a1 and S100a4, corroborating their fibrogenic fate.\nLEPR+ CD200+ were also shown to be pro-fibrotic subset of the total LEPR cells (Suhardi et al,\n2024). We have added this information into the Results and Discussion sections.\n6. Figure 4: the authors refer to the transplantation of LepR cells into a muscle pouch as an\n“osseous organoid”. Organoids are generally agreed to be three-dimensional cellular structures\nthat are derived from tissue-specific stem cells and grown in vitro. What the authors show\ninstead is a bone nodule. The histology shown is not indicative of bone, cartilage and fat\nstaining.\nWe thank the reviewer for this helpful clarification. We have revised the wording throughout the\nmanuscript to refer to the transplanted tissue as a bone nodule.\n7. Figure 6: please clarify that the anti-GREM1 antibodies were delivered daily for 2 weeks.\nPlease include data demonstrating that the anti-GREM1 used here is a function-blocking\nantibody.\nWe thank the reviewer for this comment. We have clarified in the revised figure legend and\nMethods section that the anti-GREM1 antibody was administered via twice weekly intraarticular\ninjections (highlighted in yellow in methods section). Regarding the functional characterization of\nthe antibody, we are using the same monoclonal anti-GREM1 antibody described by Chang et\nal., Nat Commun, 2019, 10,1442. In that study, the authors showed that administration of anti-\nGREM1 blocks GREM1 bioactivity, confirming its neutralizing activity.\nReviewer #2 (Remarks to the Author):\nIt is very exciting to see this kind of manuscript in the field of implantology that was dominated\nby biomechanics, engineering, and cellular biology. The vast majority of existing papers are\ndescriptive in nature.\nThis study has many strengths such as identification of therapeutic targets in the human\npathology implant loosening membranes, development of animal models with intact and mutant\nGrem1 function, neutralizing antibody treatments, and comparative transcriptomic analysis. The\nmouse animal model is rigorously analyzed by multiple IHC and quantitative microCT.\nThis study seems to be the first comprehensive mechanistic study that entailed the verification\nof Grem1 expression in LEPR+ cells in human tissue, and then replication aseptic loosening in\nclinically relevant wild type and mutant mice, therapeutic intervention using Grem1 neutralizing\nantibody, and further screening experiments for future experiments. All these features are\nexceptional strengths because literature on peri-implant fibrosis and loosening are at the cellular\ndescriptive level. Loss of Grem1 function in the context of peri-implant osseous-integration\nchanges the proliferation and tissue differentiation of the LEPR+ cells. To this end, one\nadditional ‘Figure’ (Diagram or Cartoon) that explains the mechanism of peri-implant fibrosis,\nosseous integration, and proliferation of peri-implant cells would help readers understand the\nkey concepts and therapeutic intervention. Obviously, this would not require any additional\nexperiments. In the discussion section, please add or clarify whether Grem1 is a secreted\nprotein that affects adjacent cells for differentiation into fibrous vs osteogenic cells (paracrine) or\nalters intracellular signaling for fibrous/osteogenic phenotypic changes (autocrine). These\npossible pathways could be incorporated into the Diagram. \nWe thank the reviewer for the encouraging and thoughtful comments. We are grateful for the\nrecognition of the strengths of our study, including the integration of human tissue analysis,\ngenetic mouse models, therapeutic intervention with a neutralizing antibody, and transcriptomic\nprofiling—all within the context of a clinically relevant model of aseptic loosening.\nIn response to the reviewer’s excellent suggestion, we have now added a schematic figure\n(Extended Figure 17) that illustrates the proposed mechanism of peri-implant fibrosis and\nosseous integration. This diagram incorporates our current findings and highlights the role of\nGREM1 in modulating the proliferation and differentiation of LEPR+ cells. We also included the\npossible paracrine and autocrine roles of GREM1 in directing lineage fate decisions—whether\nthrough the inhibition of BMP and Wnt pathways in adjacent cells (paracrine), or through\nmodulation of intracellular signaling in LEPR+ progenitors themselves (autocrine). We have\ndiscussed these possibilities in the revised Discussion section.\nWe thank the reviewer once again for these insightful suggestions, which have strengthened\nboth the clarity and translational relevance of the manuscript.\nIf the tissue is available for analyses, were there any difference in the number of osteoclasts?\nExcessive osteoclastic bone resorption is another mechanism of aseptic loosening. If not,\nplease add sentences in the discussion that the focus of this study is about the diversion of the\nperi-implant skeletal stem cells into fibrous tissues and osteogenic tissues. Please also mention\nthat osteoclastic bone resorption around the implant is triggered and aggravated by wear\nparticles. In the animal model that was used in this experiment, wear-particle related\ninflammation and osteoclast formation was not relevant.\nOverall, the study is outstanding by unraveling the molecular mechanism of peri-implant fibrosis\nby highly mechanistic experiments using genetic loss-of-function and neutralizing antibody in a\nclinically relevant animal model.\nWe thank the reviewer for this insightful comment regarding osteoclastic activity. In our model,\nwe examined the peri-implant tissue for Cathepsin K (CTSK)+- lineage cells (Extended Figure\n4), a marker of mature osteoclasts. We did not observe CTSK+ cells or significant in the peri-\nimplant fibrous tissue, indicating that osteoclast-mediated bone resorption was not a prominent\nfeature in this model.\nAs suggested by the reviewer, we have clarified in the Discussion section that the focus of this\nstudy is on the diversion of peri-implant skeletal stem/progenitor (LEPR+) cells toward fibrous or\nosteogenic fates, rather than on osteoclast-driven mechanisms. We also now explicitly state that\nosteoclast activity in aseptic loosening is typically associated with wear-particle–induced\ninflammation, which was not a component of the current experimental system. Our model, in\ncontrast, isolates the molecular regulation of stromal cell fate in the absence of mechanical wear\ndebris.\nWe appreciate the reviewer’s point, which has helped refine the scope and context of our work.\nReviewer #3 (Remarks to the Author):\nThe subject of the manuscript is of significant interest to the orthopaedic and dental fields since\nloosening of endoprostheses and dental implants is a great medical problem. Authors based\ntheir entire hypothesis on the idea that the major problem outside the implant is the\ndevelopment of fibrous tissue associated with leptin receptor positive cells which express\ngremlin 1, one of well known BMP antagonists. In the provided evidence authors show four\nhuman tissues collected during surgical interventions following implant loosening, and various\ngenetically modified mice following bone implants. Although authors provide a robust set of\ndata, almost the entire evidence is based on dark field fluorescence microscopy without a real\nevidence into the relationship between bone, fibrous tissue and implant surfaces. Namely, in\nFigures 3 and 6 microCT images and related bright field histology at low magnification the\npresence and separation of fibrous tissue and surrounding bone are not well visualized. In\nFigure 4b the fibrous area separating the bone from implant is outlined as red rastered area;\nhowever, in the Figure 4b Leprcre/+ mice authors use red rastered area to indicate a large area\nof fibrous tissue, under which at the implant bottom the cancellous bone is protruding beneath\nthe red, so called fibrous tissue. A similar impression exists in Figure 6b, when using a Grem1\nantibody to decrease the fibrous area. Similarly, in Figure 4e the histology does not help in\nbetter separation of fibrous tissue and bone. Since fibrous tissue on microCT images is black,\nauthors should have, instead of introducing red rastered self-drawing area, simply left the\noriginal image with fibrous tissue in originally black appearance. Since the entire further\nanalyses depend on the original well visible images, all other analyses are not convincing since\nall presentations are in dark field fluorescence, witch completely misses associated bright field\nimages to provide convincing evidence that analyzed cells and molecules are within the fibrous\ntissue and related markers stain cells, or areas of extracellular matrix outside the cells in relation\nto the implant and bone surfaces.\nWe thank the reviewer for this detailed and constructive feedback. We fully agree that clear\nvisualization of the interface between fibrous tissue, bone, and the implant surface is essential\nto support the interpretation of our findings.\nTo clarify, the microCT images shown in Figures 3 and 6 are 3D reconstructions, provided to\nhelp readers appreciate the global spatial architecture of the peri-implant tissue response.\nHowever, all quantitative analyses were performed on high-resolution 2D microCT slices, which\nallows for more precise segmentation of bone, fibrous tissue (which appears black), and the\nimplant. To address the reviewer’s concern, we have added this clarification to the Methods\nsection and included a representative 2D microCT slice and segmentation workflow in\nExtended Figure 18 to demonstrate how each component was identified and quantified.\nWe also acknowledge the importance of histological validation. In addition to H&E staining, we\nnow include Movat pentachrome, von Kossa, and plastic embedding sections to provide\ncomplementary visualization of bone, fibrous tissue, and implant interfaces. These are included\nin the revised figures and Extended Figure 8, and consistently support the tissue classifications\nused in our analysis.\nRegarding immunofluorescence, we appreciate the concern about dark field imaging. To\nenhance interpretability, we have now included the zoomed-in overlay of immunofluorescence\non the bright field images panel (Figures 2-5). These overlays improve clarity on the anatomical\ncontext of marker localization, particularly in distinguishing cellular vs. extracellular expression\nrelative to bone and implant.\nFinally, we have revised the fibrous tissue and implant masks in the figures to be more\ntransparent, allowing clearer visualization of the underlying structures.\nWe hope these additions substantially improve the clarity, transparency, and rigor of our visual\ndata presentation.\nThe mechanism indicating enrichment of the BMP pathway via analysis of relevant genes\ninvolved is not specific, since it is well known that different BMPs have different relationship with\nBMP inhibitors. It is unlikely that only Gremlin 1 exists in the fibrous area which is in tight contact\nwith the surrounding bone in which the prominent antagonist is not Gremlin, but Noggin.\nDepending on the presence of BMP2, -4, -6, -7, and -9 (maybe 12-14), Noggin inhibits BMP2, -4\nand -7, while BMP6 and BMP9 escape inhibition by Noggin due to one aminoacid difference\n(lysin) in the position 60 of the N terminal part of the molecule. Authors mention that Gremlin\ninhibits BMP2 and -7, but did not mention its effect on remaining BMPs, neither analyzed the\nfibrous tissue for specific BMPs and their inhibitors to make their mechanistic proposal fully reliable. It might be that Noggin is present as well in the fibrous tissue, including BMP6 or\nBMP9, which makes then the enrichment of BMP pathway analysis less convincing.\nWe thank the reviewer for this insightful and knowledgeable comment highlighting the\ncomplexity of BMP signaling and the specificity of interactions between BMP ligands and their\nantagonists. We fully agree that the differential binding affinities of BMP inhibitors—such as\nGremlin 1 and Noggin—to various BMPs are critical in understanding pathway modulation in the\nperi-implant environment. As noted by the reviewer, Noggin inhibits BMP2, BMP4, and BMP7,\nwhile BMP6 and BMP9 are resistant to Noggin inhibition due to structural differences.\nGremlin 1 is known to antagonize BMP2, BMP4, and BMP7, with variable affinity.\nTo address this point, we analyzed the expression of key BMP ligands and inhibitors in the\nperi-implant tissue using both bulk RNA-seq and immunofluorescence. Our data show that\nconditional deletion of Grem1 in LepR-lineage cells results in upregulation of Bmp4 and\nBmp7, but not Bmp2, Bmp6, Bmp9, or Noggin (Extended Data Fig. 11-13). While we\nacknowledge that other inhibitors such as Noggin may be expressed in the peri-implant\nenvironment, our transcriptomic data did not show significant upregulation of Nog in the\nfibrous tissue, and we did not detect robust protein-level Noggin expression in this context.\nBased on these findings, we propose that the enhancement of BMP signaling observed\nfollowing Grem1 deletion is likely driven by increased availability of BMP4 and BMP7,\nwhich are normally suppressed by GREM1 but not by Noggin, thus reinforcing the specificity\nof GREM1's role in modulating this signaling axis in LEPR-lineage cells.\nWe have expanded the Discussion to acknowledge the broader BMP signaling context and\nadded these additional details to strengthen the mechanistic interpretation (highlighted in\nyellow).\nSpecific issues:\n1. In Abstract authors claim that 18 million dental implants and 2 million total joint implants are\nplaced annually. However, in Introduction they suddenly claim 18 million dental implants and 1.3\nmillion total joint implants.\nWe thank the reviewer for pointing this out. We have revised the manuscript to ensure\nconsistency between the Abstract and the main text regarding implant procedure statistics.\n3. In 1g scale bar in the text of 250 micrometers is 500 micrometers. In 1e and 1k a similar\ndescription is repeated, while e refers to human and k to mouse.\nWe thank the reviewer for noting these inconsistencies. We have corrected the scale bar label in\nFigure 1g to reflect the accurate measurements and have revised the figure legends and\nassociated text to ensure consistent and accurate descriptions across panels 1e and 1k. These\ncorrections have been incorporated into the revised manuscript and figure files.\n4. In Figure 2 different graphs quantifying the colocalization refer to different numbers of\nsamples and claim that they represent three independent experiments. This should be clarified,\nsince it is not clear how many samples are from each of three experiments included in the analysis.\nThank you for your comment, all biological replicates from three separate experiments are\npooled and included in the analysis.\n5. Figure 4 presents in e and j data on bone organoids, which are of low imaging quality.\nThank you for the comment, we have provided a higher resolution image to enhance readability.\n6. Instead of staining bone and fibrous tissue with hematoxylin, the bone should have been\npresented by von Kossa mineral stain (black) which would eventually separate bone and fibrous\ntissue.\nWe appreciate the reviewer’s suggestion regarding the use of von Kossa staining to improve the\ndistinction between mineralized bone and fibrous tissue. In response, we have performed von\nKossa staining and included the results as Extended Figure 8. Quantitative analysis using von\nKossa confirmed that the percentage of bone and fibrous tissue is consistent with\nmeasurements obtained from both H&E and Movat’s pentachrome staining. These findings\nvalidate the robustness of our histomorphometric quantification across multiple staining\nmethods. We have incorporated this results in the Results section (highlighted in yellow).\n7. Instead of BMP pathway gene analysis, which seems less relevant for this study, a structure\nfunction analysis of BMPs vs. Gremlin 1 would have been much more helpful to demonstrate\ninhibition of BMP6 as well, which might be present in adjacent bone, escaping the Noggin\ninhibition.\nWe thank the reviewer for this insightful suggestion. To explore the functional link between\nGREM1 and BMP signaling in the peri-implant environment, we performed both transcriptomic\nanalysis and immunofluorescence staining of the peri-implant region. We found that\nconditional deletion of Grem1 in LepR-lineage cells resulted in upregulation of Bmp4 and\nBmp7, but not Bmp2, Bmp6, Bmp9, or Noggin (Extended Figure 10-12). This is in line with\nprior findings that GREM1 binds to BMP4 and BMP7.1,2\nBased on these findings, we propose that GREM1 inhibition promotes osteogenesis in part\nthrough the activation of BMP signaling mediated by BMP4 and BMP7. Upregulation of\nBMP pathways in turn resulted in redirection of LEPR-lineage cells away from a fibrogenic fate\nand toward osteogenic differentiation.\n8. The majority of small letters in figures throughout the manuscript are too small and not\nreadable, see extended Figure 7 as an example.\nWe thank the reviewer for the input regarding improving readability. We have increased the font\nsize while adhering to the figure preparation guidelines for Nature Communications. We have\nalso provided a higher resolution image to improve text clarity.\nReviewer #4 (Remarks to the Author):\nGeneral comments: The MS by Suhardi et al reports that exuberant expression of GREM1 by\nLepr+ cells drives fibrosis and the expense of osteogenesis at sites of implant placement into\nbone and that reducing GREM1 via local injection of neutralizing antibody reduces fibrosis and\nenhances osteogenesis. As peri implant fibrosis is a significant clinical problem and treatment\nwith GREM1 neutralizing antibody is a simple clinical procedure, these findings may be of high\nimpact. However, as presented the data raise concerns about the variable nature of the\nresponse to GREM1 antibody treatment in mice (see below for specifics) and that, coupled with\nthe small number of human samples assayed for the presence of GREM1 (n=4 with a wide age\nrange) at sites of peri implant fibrosis, raise concerns about the therapeutic potential of these\nfindings. Additionally, the authors published a very similar finding in 2024 by targeting ADGRF5\nin the same Lepr+ cell population but do not compare their new findings to these already\npublished studies, raising concerns about the significance of targeting any one gene product as\na therapeutic approach to peri-implant fibrosis and reducing the overall novelty of the current\nstudy.\nWe thank the reviewer for their thoughtful and constructive comments. We appreciate the\nrecognition of the clinical relevance and potential impact of targeting GREM1 to mitigate peri-\nimplant fibrosis and enhance osseointegration.\nIn response to the concern regarding human data, we have now increased the number of\npatient samples analyzed. In this expanded cohort, all patients with aseptic loosening\nconsistently exhibited abundant LEPR⁺GREM1⁺ cells within the peri-implant fibrous membrane,\nreinforcing the therapeutic relevance of targeting GREM1 in the human disease context. These\nfindings are now reflected in the Methods section and updated figure panels (Extended Figure\n2).\nRegarding the novelty and distinction from our previous work on ADGRF5, while both studies\ntarget LEPR-lineage stromal cells, the current manuscript describes a mechanistically distinct\nand therapeutically independent pathway. Our prior publication (Suhardi et al., 2024) focused on\nADGRF5 and its role in stem cell regulation within the bone marrow niche. In contrast, this study\ndemonstrates that GREM1 acts through a separate and independent regulatory axis, as shown\nin Extended Figure 13, where conditional deletion of Grem1 does not affect ADGRF5\nexpression or signaling, and vice versa. Furthermore, Grem1 deletion leads to robust activation\nof BMP and Wnt signaling pathways, which are not altered in the Adgrf5 mutant model—\nhighlighting non-overlapping downstream mechanisms.\nTaken together, these findings establish that GREM1 and ADGRF5 represent distinct molecular\nlevers for modulating peri-implant stromal cell fate. The current study is the first to identify\nGREM1 as a regulator of peri-implant fibrosis and to demonstrate that its inhibition via a\nneutralizing antibody can therapeutically redirect cell fate toward osteogenesis, even in an\nestablished fibrotic environment. This expands the therapeutic landscape by offering an\nalternative and potentially synergistic strategy for promoting implant integration.\nWe have revised the Introduction and Discussion to better highlight the added value and novelty\nof the current study relative to our prior work, as suggested.\nSpecific comments:\n1. Line 85: there seems to be a sentence missing about the identification of GREM1.\nThank you for the comment, we have added the missing sentence.\n2. Materials and methods: there is a description of isolation of mesenchymal cells from bone,\nbut it is unclear if the cells retrieved include a heterogeneous population including periosteal\ncells, trabecular and cortical bone osteoblasts and bone marrow. As the number and types of\nLepr+ cells would change as a function of age and health status in patients, it is unclear what\nthis mixed population is a model of.\nWe thank the reviewer for this important observation. In this study we focused our analysis on\npro-osteogenic LEPR⁺ cells that co-express BGLAP, OSX, and GREM1 (Fig. 2), and their\ncapacity to differentiate into a pro-fibrotic LEPR⁺CD200⁺ phenotype under pathological\nconditions. The LEPR⁺CD200⁺ signature has been previously described as marking a pro-\nfibrotic subset of LEPR cells (Suhardi et al., 2024). We have clarified this focus in the revised\nMaterials and Methods and Discussion sections to better reflect the scope and intent of our\ncellular analysis.\n2. Figures would benefit from inclusion of a representative histology section that allows the\nreader to orient to the immunolocalizations. It is hard to gauge how representative the areas\nfocused on in the panels are in relation to the actual overall histology.\nWe thank the reviewer for this helpful suggestion. In response, we have added the\nimmunofluorescence-bright field overlay where to Figure 2,3,4, and 5.The updated figures and\nlegends have been revised accordingly in the main manuscript.\n3. Discussion:\na) The authors suggest that Lepr+ cells need to initiate osteogenesis to produce GREM1 and\nstate that GREM1 expression blocks osteogenesis. It isn't clear from this model why GREM1\nwould be constantly on if osteogenesis is not occurring. No mention is made about what\nspecifically regulates GREM1 expression in the Lepr+ cells. It is also unclear if all Lepr+ cells\nhave equal ability to become osteoblasts and produce GREM1.\nWe appreciate the reviewer for this insightful comment. In our study, we observed that GREM1\nexpression is significantly upregulated in the LEPR-lineage cells under fibrogenic conditions\nrelative to a baseline control (Figure 2). Importantly, GREM1 expression emerges after the\ninitial expression of early (OSX) and late (BGLAP) osteogenic markers in LEPR+ cells (Figure\n2), suggesting that LEPR+ cells transiently adopt an osteogenic transcriptional program before\nacquiring a pro-fibrotic phenotype marked by CD200 and high GREM1. This is consistent with a\nmodel in which LEPR+ cells with initial osteogenic tendency being diverted into a fibrotic fate,\nmediated in part by autocrine expression of GREM1.\nAs shown in Figure 2m–r, GREM1 expression increases progressively from postoperative day\n3 to 14, with strong spatial correlation to LEPR+ cells expressing BGLAP. This supports the\nmodel that GREM1 expression is temporally downstream of osteogenic commitment but\nprecedes terminal osteoblast differentiation. Furthermore, our BrdU experiments (Figure 4) and\nengraftment data (Figure 4e–p) show that deletion of Grem1 reduces proliferation and restores\nosteogenic differentiation capacity, suggesting that GREM1 functions in an autocrine loop to\nsuppress osteogenesis and maintain a fibrotic phenotype.\nb) No mention is made about how stable the rescue of osteogenesis is with injection of the\nGREM1 antibody. How does the antibody disrupt the feedback loop between osteoblast\ndifferentiation and GREM1 production and what happens when the antibody is no longer\npresent as would be the case for treating patients?\nWe appreciate the reviewer’s question regarding the durability of the osteogenic rescue\nfollowing GREM1 neutralization. Our prior work (Suhardi et al., Nat Biomed Eng, 2024, 8:1285-\n1307) demonstrated that the peri-implant cellular composition stabilizes by two weeks post-\nsurgery in the absence of additional perturbation. Based on this, we anticipate that redirection of\npro-fibrotic LEPR⁺ cells toward an osteogenic lineage by anti-GREM1 antibody results in the\nestablishment of bone at the implant interface, which in turn eliminates micromotion—a primary\nstimulus for peri-implant fibrosis.\nBy neutralizing GREM1, which functions as a key autocrine inhibitor of BMP signaling, the\nantibody disrupts the positive feedback loop that maintains LEPR⁺ cells in a proliferative, pro-\nfibrotic state and redirection toward osteogenic differentiation. This process leads to the\nformation of a mineralized, stable peri-implant interface. Once this interface is established and\nmicromotion ceases, the fibrogenic trigger is removed, and the newly formed bone is maintained\nin the absence of ongoing antibody therapy.\nWe have added a section to the revised Discussion elaborating on the expected durability of the\nosteogenic response and its relationship to implant stability and the fibrotic niche (highlighted in\nyellow).\nc) The authors state that both the BMP and Wnt pathways are enhanced when GREM1 is\nblocked but do not discuss which of these signals is required for the reinitiating of osteogenesis.\nWe thank the reviewer for raising this important point. Both BMP and Wnt signaling pathways\nare upregulated following GREM1 deletion or neutralization, as shown in our transcriptomic,\npSMAD 1/5/8 and activated beta-catenin analyses (Figure 5 and Extended Figure 10-12).\nBMP signaling and Wnt/β-catenin signaling are required to initiate and maintain bone\nregeneration (Tsuji et al., Nat Genet, 2006, 38:1424–1429, Minear et al., Sci Transl Med, 2010,\n2:29ra30).\nIn our study, GREM1 inhibition led to the upregulation of both pathways, suggesting that its\nblockade relieves a key inhibitory checkpoint and increase LEPR+ cells to be pro-osteogenic.\nWe have now added a section to the Discussion highlighting the respective roles of BMP and\nWnt signaling in reinitiating osteogenesis following GREM1 inhibition.\nd) The authors do not address how current findings with blocking GREM1 compare to those\nthey reported in 2024, in the same animal model, of blocking ADGRF5. As the authors claimed\nanti ADGFR5 therapy was efficacious, it is incumbent upon them to show why additional therapy\nto block GREM1 is necessary and provides better results than blocking ADGFR5\nWe thank the reviewer for raising this important point. While both our previous study on Adgrf5\n(Suhardi et al., 2024) and the current manuscript target LEPR⁺ skeletal stem/progenitor cells,\nthe mechanistic pathways, biological functions, and translational implications are fundamentally\ndistinct and independent.\nIn the present study, we identify Grem1—a secreted antagonist of BMP signaling—as a key\nupstream regulator of LEPR⁺ cell proliferation and lineage specification. We demonstrate that\nGrem1 and Adgrf5 function independently, as conditional deletion of one does not alter the\nexpression or signaling activity of the other (Extended Figure 13). While downregulation of\nGrem1 resulted in upregulation of BMP and WNT pathways, downregulation of Adgrf5 did not\naffect the BMP nor WNT pathways (Extended Figure 13).\nFrom a translational perspective, GREM1 neutralizing antibodies have already undergone\npreclinical testing and shown favorable safety profiles (Rivera IR et al., Cancer Res, 2024;\nSarker D et al., Cancer Res, 2023). These prior studies provide a foundational safety and\nefficacy framework that supports the clinical potential of GREM1 inhibition for the treatment of\nperi-implant fibrosis. More generally, given the expected extremely high attrition rate of moving\ntherapeutic candidates from preclinical mechanistic studies to an approved real world clinical\npractice, we believe that it would be greatly premature to consider any ADGRF5 targeting\ntherapy as pre-empting the GREM1 targeted therapy proposed here until ADGRF5-based\ntherapies are actually established as a standard of care.\nThus, our current findings not only uncover a novel and mechanistically distinct pathway\nunderlying peri-implant fibrosis but also highlight GREM1 as a promising therapeutic target with\nexisting translational momentum. We have revised the Discussion section to clearly articulate\nthese distinctions and have included relevant references to prior work on GREM1-targeted\ntherapies (highlighted in yellow).\n12/20/2025\nRe: Manuscript NCOMMS-25-07110 “GREM1 acts in leptin receptor-expressing skeletal cells to\nmediate peri-implant fibrosis”\nPoint-by-point response\n(Editor and reviewer comments are in red, our response is in black)\nWe thank the Editor and Reviewers for their careful evaluation of our manuscript and for their\nconstructive and thoughtful feedback throughout the review process. We are grateful for the\nreviewers’ recognition of the conceptual rigor, mechanistic depth, and translational relevance of\nthis work. Below, we provide our final point-by-point responses.\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe revised paper package includes (1) thorough conceptual and technical responses to\ncritiques; and (2) revised manuscript.\nThe central hypothesis, results, and interpretation of data are very logical.\nThis revised manuscript is very much acceptable and will be of great interest to readers with\nrespect to the first time mechanistic explanation of peri-implant fibrosis and possible therapeutic\nconversion/prevention of fibrosis towards peri-implant osteogenesis.\nWe sincerely thank Reviewer #2 for this positive and encouraging assessment. We are grateful\nfor the recognition that this work provides a first mechanistic framework for peri-implant fibrosis\nand demonstrates a therapeutically actionable strategy to redirect fibrosis toward osteogenesis.\nNo additional revisions were requested, and we appreciate the reviewer’s strong support for\npublication.\nReviewer #3 (Remarks to the Author):\nThe authors have done additional work to respond to the criticism of the original submission.\nIt is now more convincing that GREM1 has a role in the peri implant fibrosis. Avoiding to\nmention their previous publication with the same model and peri implant fibrosis in association\nwith another molecule ADGRF5 was a significant mistake although it is now explained that the\nsame positive effect via inhibition of both of them uses different pathways is a very delayed\nresponse to the effect the authors try to explain. However, there is now few paragraphs dealing\nwith this issue. Other issues have been corrected and the majority of figures substituted with\nbetter once while several have been added to the supplementary material. Overall, the\nmanuscript is significantly improved. It also grew in number of pages, experiments and figures\nwhich makes it very rich in the major results and additional experimental descriptions and\ndescriptions of figures. It takes quite a bit of time to read it and digest its reliability, when\ncompared to the original submission.\nI would suggest to publish these results in their revised version.\nWe thank Reviewer #3 for their careful reassessment and for acknowledging the substantial\nstrengthening of the manuscript. We appreciate the emphasis on properly contextualizing the\npresent findings relative to our prior work on ADGRF5.\nIn the final manuscript, we explicitly address this point in the Introduction and Discussion. While\nboth ADGRF5 and GREM1 act within the same LEPR⁺ skeletal progenitor population and both\nreduce peri-implant fibrosis when inhibited, our data demonstrate that these pathways are\nmechanistically independent. Conditional deletion of Grem1 does not alter ADGRF5 expression\nor signaling, and conversely, deletion of Adgrf5 does not affect GREM1 levels. Importantly,\nGREM1—but not ADGRF5—functions as a dominant brake on BMP4/BMP7 and WNT/β-catenin\nsignaling in the peri-implant niche. These distinctions are supported by genetic, transcriptomic,\nand signaling analyses presented in the final manuscript.\nWe appreciate the reviewer’s recognition that the expanded experiments, figures, and\nsupplementary analyses improve both the robustness and clarity of the study, and we thank\nthem for recommending publication.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my scientific concerns. There are several minor clarifications that\nwould improve the MS.\n1. Line 72: adding the fact that modulating the ADGRF5 pathway also reduces fibrosis and\nenhances osteogenesis would help to clarify this sentence.\nResponse:\nThe manuscript now explicitly states that modulation of ADGRF5 signaling reduces peri-implant\nfibrosis and enhances osteogenesis, while emphasizing that the current study identifies GREM1\nas a mechanistically distinct pathway.\n2. Line 75: the \"mechanistic independence\" is in comparison to ADGRF5 so that needs to be\nstated.\nResponse:\nThis clarification has been incorporated. The text now clearly specifies that mechanistic\nindependence is in comparison to the ADGRF5 pathway.\n3. Line 183: I do not believe that unsurgerized is an actual word so perhaps saying control tibiae\nor tibiae that did not undergo surgery would be better.\nResponse:\nThis term has been replaced with “control tibiae that did not undergo surgery.”\n4. Line 225: No protein analyses were performed so it is more accurate to state that mRNA\nlevels for BMPs were changed.\nResponse:\nThe manuscript now accurately states that changes were observed at the mRNA level, as no\nprotein-level analyses were performed for these BMP ligands."}]

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